Micronized placental compositions comprising a chelator

ABSTRACT

Provided herein are micronized placental compositions composed of micronized placental tissue component, such as amnion or chorion and/or filler bound to one or more chelating agents, which in turn are optionally bound, reversibly, to pharmacologically active metal ions. Further provided are methods of making and using the placental compositions. The compositions have numerous therapeutic applications.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit under 35 U.S.C. § 119(e) of U.S.Provisional Application No. 61/872,393, filed on Aug. 30, 2013, which isincorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

Field of the Invention

This invention is directed to micronized placental compositionscomprising micronized placental components and optionally a filler,wherein a biologically compatible chelator moiety is conjugated to acomponent of the composition. This invention further comprises suchcompositions having pharmacologically active metal ions reversibly boundto the chelating moiety.

State of the Art

Placental tissue components such as isolated amnion and chorion as wellas laminates thereof are known in the art for use as wound coverings andto promote wound healing. Typically, placental tissue is harvested afteran elective Cesarean surgery. The placenta is composed of an amnioticmembrane which has two primary layers of tissue, amnion and chorion.Amnion tissue is the innermost layer of the amniotic sac and in directcontact with the amniotic fluid. The amniotic sac contains the amnioticfluid and protects the fetal environment. Histological evaluationindicates that the membrane layers of the amnion consist of a singlelayer of epithelium cells, thin reticular fibers (basement membrane), athick compact layer, and a fibroblast layer. The fibrous layer of amnion(i.e., the basement membrane) contains collagen types IV, V, and VII,and cell-adhesion bioactive factors including fibronectin and laminins.The amnion so recovered is commercially used in wound grafts whichprotect the wound and induce healing.

In addition to such beneficial uses, amnion has been found to act as astem cell recruiter as provided in U.S. Patent Application PublicationNo. 2014/0106447, which is incorporated herein by reference in itsentirety.

U.S. Patent Application Publication 2013/0344162 describes micronizedplacental tissue compositions and methods of making and using the same.U.S. patent application Ser. No. 13/903,878 describes biologicallycompatible polymer-chelator conjugates and methods of making and usingthe same. These references are incorporated herein by reference in theirentirety.

Koob et al. have described methods of producing nordihydroguaiareticacid (NDGA) polymerized and cross-linked collagen fibers for variousbiomedical applications, some with tensile strengths similar to that ofnatural tendon (e.g., about 91 MPa). See, for example, Koob andHernandez, Material properties of polymerized NDGA-collagen compositefibers: development of biologically based tendon constructs,Biomaterials 2002 January; 23 (1): 203-12; and U.S. Pat. No. 6,565,960,the contents of which are hereby incorporated by reference as if recitedin full herein.

SUMMARY OF THE INVENTION

This invention is directed to compositions comprising micronizedplacental tissue components, such as amnion, chorion, intermediatetissue layer and Wharton's jelly, as well as laminates thereof, andoptionally a filler, wherein at least a portion of the placentalcomponents and/or the filler is conjugated to chelating moieties.Preferably, the chelating moieties have releaseably boundpharmacologically active metal ions such that the metal ions arereleased in a sustained manner over time. The actions of these metalions can thereby be coupled with the therapeutic effects of micronizedamnion in a localized manner to provide enhanced therapy.

Alternatively, the compositions of this invention can be used to kill ordisrupt aberrant cells including aberrant stem cells. In such anembodiment, the compositions comprise placental components, such asamnion and chorion as well as laminates thereof, and optionally afiller. Either or both are coupled to a chelating agent havingreversibly bound thereto pharmacologically active agent comprising oneor more pharmacologically active metal ions such as cisplatin. In someembodiments, the amnion is used to recruit the aberrant stem cells suchas cancer stem cells (CSCs) and the anti-cancer agents are used to killor disrupt the CSCs. In this regard, CSCs are believed to be responsiblefor cancer recurrence, initiation, progression, metastasis, and drugresistance. Without being bound by theory, it is believed thatintroduction of a composition of the current invention at the site of atumor will result in targeting of the CSCs by a metal-containinganticancer agent. That is to say, the CSCs will be recruited to thetumor site by the amnion and will be killed or inhibited by theanticancer agent.

In one of its composition aspects, the invention relates to acomposition comprising micronized placental tissue and componentsthereof such as amnion, chorion and laminates thereof and optionally afiller wherein at least a portion of the micronized tissue and/or filleris conjugated to one or more chelating moieties. In addition, micronizedumbilical cord materials such as micronized Wharton's jelly can becombined with the micronized placental tissue.

In another of its composition aspects, therapeutically active metal ionsare releasably bound to the chelating moieties. In one aspect, the metalions are anticancer agents which are released over time so as to providesustained anticancer activity.

The placental composition, such as amnion, chorion, intermediate tissuelayer, Wharton's jelly composition, of the invention may be introducedin a patient having cancer that is amenable to treatment with a metallicion, for example, platinum alkylators. Such compositions may beintroduced into the patient before, during, and/or after treatment ofthe tumor. In one aspect, the composition is introduced at or near thesite of a tumor that is otherwise not treatable, for example, aninoperable brain tumor or spinal cord tumor. In one aspect, thecomposition is introduced at or near the site of a tumor in conjunctionwith other treatment, for example surgery, chemotherapy, and/orradiation therapy. In one aspect, the composition is introduced at ornear the site of a tumor after the tumor has been treated and/orremoved.

The chelating moiety can be any moiety with functional groups pendentthereto which are suitable for reversibly binding biologicallycompatible and pharmacologically active metal ions. Examples of suchbiologically compatible and pharmacologically active metal ions include,without limitation, ions of silver, copper, or a metallic ionicanti-tumor agent. Preferably, the anti-tumor agent is ionic platinum.The chelating moieties described herein can contain a mixture of metalssuch as anti-bacterial silver ions and/or anti-fungal copper ions toinhibit opportunistic bacterial and/or fungal infections at the sitetreated for cancer.

In one embodiment, the placental composition, such as amnion or chorioncomposition of the invention, is injected at or near the affected area.In one embodiment, the placental composition is implanted at or near theaffected area. Without being bound by theory, it is believed thatlocalized introduction of the composition reduces the potential sideeffects that occur with systemic treatment with pharmacologically activemetal ions.

This invention is also directed to methods for making and using thecompositions described herein having one or more chelating moietiesbound thereto.

Several of the advantages of this invention are set forth in part in thedescription which follows, and in part will be obvious from thedescription, or may be learned by practice of the aspects describedbelow. The advantages described below will be realized and attained bymeans of the elements and combinations particularly pointed out in theappended claims. It is to be understood that both the foregoing generaldescription and the following detailed description are exemplary andexplanatory only and are not restrictive.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawing(s), which are incorporated in and constitute apart of this specification, illustrate several aspects described below.

FIGS. 1A and 1B are overview flow charts of the process for making theamnion compositions described herein.

FIGS. 2 and 3 illustrate different binary release patterns of metallicions from a composition comprising a mixture of amnion- orfiller-chelating agent conjugates using different chelating moieties. InFIG. 2, the release rates of the metal ions from a chelating agentconjugate containing different non-cross-linking chelating agents(moieties) are illustrated. The release rates from these two differentchelating agents are selected to overlap so as to provide a sustainedrelease of the metal ion over time. In FIG. 3, the release rates of themetal ions from two similar conjugates are illustrated. In this case,the chelating moieties of the conjugates are selected so that theirrelease rates do not to overlap. This results in release of a firstbolus of metal ion release followed by release of a second bolus ofmetal ion.

FIG. 4 shows a schematic for a cell culture insert for stem cellmigration assays described in Example 3.

FIG. 5 shows a bar graph of percent cell migration in human mesenchymalstem cells (MSCs) cultured in the presence of various amounts ofEpiFix®. Details are described in Example 3.

FIG. 6A shows a bar graph of percentage living/Lin⁻ mouse hematopoieticstem cells in normal skin, sham implant, acellular dermal matrix, andEpiFix® at 3, 7, 14, and 28 days post implant. Values shown aremeans+/−standard deviation, n=4 specimens. ** indicates p<0.05 whencomparing EpiFix® or control ADM to normal skin and sham implant viaone-way ANOVA. †† indicates p<0.05 when comparing EpiFix® to control ADMvia two tailed t-test. FIG. 6B shows a bar graph of percentageliving/Lin⁻ mouse mesenchymal cells in normal skin, sham implant,acellular dermal matrix, and EpiFix® at 3, 7, 14, and 28 days postimplant. Values shown are means+/−standard deviations, n=4 specimens. **indicates p<0.05 when comparing EpiFix® or control ADM to normal skinand sham implant via one-way ANOVA. Details are described in Example 4.

FIG. 7A shows representative FACS dot plots of cells detected using flowcytometry and fluorescent detection of CD45 and Sca-1. FIG. 7B showsphotomicrograph of dermal tissue stained with DAPI which stains cellbodies, and CD34, which is a marker for hematopoietic stem cells.Details are described in Example 5.

DETAILED DESCRIPTION

Before the present invention is disclosed and described, it is to beunderstood that the aspects described below are not limited to specificcompositions, methods or preparing such compositions, or uses thereof assuch may, of course, vary. It is also to be understood that theterminology used herein is for the purpose of describing particularaspects only and is not intended to be limiting.

In this specification and in the claims that follow, reference will bemade to a number of terms that shall be defined to have the followingmeanings:

All numerical designations, e.g., pH, temperature, time, concentration,and weight, including ranges of each thereof, are approximations thattypically may be varied (+) or (−) by increments of 0.1, 1.0, or 10.0,as appropriate. All numerical designations may be understood as precededby the term “about”.

It must be noted that, as used in the specification and the appendedclaims, the singular forms “a,” “an” and “the” include plural referentsunless the context clearly dictates otherwise. Thus, for example,reference to “a bioactive agent” includes mixtures of two or more suchagents, and the like.

“Optional” or “optionally” means that the subsequently described eventor circumstance can or cannot occur, and that the description includesinstances where the event or circumstance occurs and instances where itdoes not. For example, the phrase “optionally cleaning step” means thatthe cleaning step may or may not be performed.

The term “amnion” as used herein includes amniotic membrane where theintermediate tissue layer is intact or has been substantially removed.

The term “amnion composition” refers to a composition comprising one ormore placental tissue components wherein at least one of the componentsis amnion. The amnion composition optionally includes a filler. The termamnion composition further optionally includes one or more chelatingagents bound to a component(s) of the composition.

The term “chorion composition” refers to a composition comprising one ormore placental tissue components wherein at least one of the componentsis chorion. The chorion composition optionally includes a filler. Theterm chorion composition further optionally includes one or morechelating agents bound to a component(s) of the composition.

The term “filler” refers to any component of the composition other thanamnion. Filler includes other placental tissue components as well aspolymers, including collagen, hyaluronic acid, biocompatibleplasticizers and the like. In one embodiment, the collagen include humancollagen and collagen prepared from placental tissue, such as collagenmaterials substantially free of non-human antigens. In some embodiments,the collagen is prepared form the fibrous layer of amnion (i.e., thebasement membrane) which contains collagen types IV, V, and VII. Thecollagen filler described herein is separate and apart from thecollagen, if any, that exists in the placental component, such as in theamnion or chorion, of the composition. In some aspects, the collagen isfree or substantially free of other components, including elastin,fibronectin, and/or laminin.

The term “non-human antigen” refers to any agent (e.g., protein,peptide, polysaccharide, glycoprotein, glycolipid, nucleic acid, orcombination thereof) of non-human origin that, when introduced into ahuman, is immunogenic, eliciting an unwanted immune response thatrequires medical treatment for the manifestations (e.g., inflammation,etc.) of the immune response. As defined herein, the non-humanantigen-induced immune response can be humoral or cell-mediated, orboth.

The term “placental tissue” refers to any and all of the well-knowncomponents of the placenta including but not limited to amnion, chorion,intermediate tissue layer, umbilical cord component, such as Wharton'sJelly, and the like. In one embodiment, the placental tissue does notinclude any of the umbilical cord components (e.g., Wharton's jelly,umbilical cord vein and artery, and surrounding membrane). In anotherembodiment, the placental tissue includes any of the umbilical cordcomponents (e.g., Wharton's jelly, umbilical cord vein and artery, andsurrounding membrane).

The term “mold,” “molded,” or “molding” includes any form of moldingsuch as the use of actual molds, extrusion under pressure, stamping orany pressurized method, and the like such that micronized placentaltissue, and optionally filler, are compressed under pressure to producea placental composition that has a defined size and shape for a definedperiod of time for use either ex vivo or in vivo. The molded placentalcomposition preferably has a sufficient density and cohesiveness tomaintain its size and shape during administration in vivo.

The term “comprising” means any recited elements are necessarilyincluded and other elements may optionally be included. “Consistingessentially of” means any recited elements are necessarily included,elements that would materially affect the basic and novelcharacteristics of the listed elements are excluded, and other elementsmay optionally be included. “Consisting of” means that all elementsother than those listed are excluded. Embodiments defined by each ofthese terms are within the scope of this invention.

“C_(m)” when placed before a group refers to that group containing mcarbon atom(s).

“Alkyl” refers to a hydrocarbyl radical, preferably monovalent,containing 1-12 carbon atoms. Non limiting examples of alkyl includesmethyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl, andthe like.

“Cycloalkyl” refers to a cyclic hydrocarbyl radical, preferablymonovalent, containing 3-10 carbon atoms and includes bicyclic radicals.Non-limiting examples of cycloalkyl include cycloproyl, cyclobutyl,cyclopentyl, cyclohexyl, and the like.

“Aryl” refers to an aromatic hydrocarbyl mono-, bi-, or tricyclic ringshaving from 6 to 14 carbon atoms.

“Heteroayl” refers to an aryl ring containing 1-5 ring heteroatomsselected from nitrogen, oxygen, sulfur, and appropriate oxidized formsthereof and having from 1 to 14 carbon atoms.

“Aldehyde” refers to a compound of formula O═C(H)—R wherein R isselected from the group consisting a chelating moiety connected to thealdehyde optionally through a linker moiety. When a linker is employed,the aldehyde is represented by the formula R-L-aldehyde and L is thelinker.

“Isocyanate” refers to a compound of formula O═N═C-L-R where R is asdefined above and L is a bond or a linker moiety.

The term “linker” defined above as “L” refers to a covalent bond or alinking group having 1 to 10 atoms selected from oxygen, sulfur,nitrogen and carbon atoms which links the chelating moiety to theplacental component, such as amnion or chorion and/or the filler.

The term “chelating moiety” refers to well-known substituents whichcontain functional groups capable of chelating metal ions. Suchsubstituents include by way of example only α, β groups such as hydroxylgroups, carboxyl groups, amino groups, or mixtures thereof (a carboxyland a hydroxyl group). The chelating moiety reversibly bonds to themetal ion and the strength of the chelation is measured by adissociation constant. The art is replete with different dissociationconstants for binding different metal ions to specific and wellcharacterized chelating moieties.

The term “subject” or “patient” as used herein refers to any vertebrateorganism including, but not limited to, mammalian subjects such ashumans, farm animals, domesticated pets and the like.

The term “biocompatible” as used herein refers to a material that issuitable for implantation or injection into a subject. In variousaspects, a biocompatible material does not cause toxic or injuriouseffects once implanted in the subject.

The term “modified placental tissue” refers to any and all components ofplacental tissue including whole placental tissue that has been modifiedby cleaning, disinfecting, and/or segmenting the tissue as well as toseparated components of placental tissue such as amnion, chorion, theumbilical cord, and the like. Modified tissue may maintain cellularlayers, such as the epithelial layer and/or the fibroblast layer.Modified placental tissue may include further modification, such aslamination of one or more layers of placental tissue, micronization ofplacental tissue, chemisorption or physisorption of small molecules,proteins (e.g. growth factors, antibodies), nucleic acids (e.g.aptamers), polymers, or other substances.

The term “sufficient amount of” refers to an amount of a compositionthat is sufficient to have the desired effect, e.g. provoke stem cellrecruitment proximate to or on the composition over time, either in vivoor in vitro. The “sufficient amount” of an placental composition, suchas an amnion or chorion composition will vary depending on a variety offactors, such as but not limited to, the type and/or amount of placentalcomposition used, the type and/or amount of filler used, the type and/orsize of the intended organ and/or body part to be treated, the severityof the disease or injury to the organ and/or body part to be treated andthe administration route. The determination of a “sufficient amount” canbe made by one of ordinary skill in the art based on the disclosureprovided herein.

As used herein, an “anticancer amount of metal ion” or “anticanceramount of anticancer agent” refers to an amount of anticancer agent,such as a metal ion, chelated to a composition of this invention thatwhen contacted in vitro or in vivo with aberrant cells, for examplecancer cells, inhibits or kills aberrant cells or tissue, and inhibitsor kills, preferably, 50%, more preferably, 90%, and still morepreferably 99% of such cells or tissue. As used herein, “effectiveamount of anticancer agent” refers to an amount of anticancer agent thatis, preferably, released from a composition of this invention and thatis sufficient to inhibit or kill, in vitro or in vivo, preferably, 50%,more preferably, 90%, and still more preferably 99% of aberrant cells ortissue. An effective amount of anticancer agent can improve one or morecancer symptoms, and/or ameliorate one or more cancer-side effects,and/or prevent and/or impede invasiveness and/or metastasis of cancer.In certain embodiments, an anticancer amount of anticancer agent candiffer from an effective amount of anticancer agent.

The term “stem cell recruiting factors” refers to any and all factorsthat are capable of recruiting stem cells and causing them to migratetowards a source of such factors. Non-limiting examples of stem cellrecruiting factors may be one or more CC chemokines, CXC chemokines, Cchemokines, or CX3C chemokines.

The term “stem cell recruitment” refers to direct or indirect chemotaxisof stem cells to a placental composition, such as an amnion or chorioncomposition. The recruitment may be direct, wherein stem cell recruitingfactors (e.g. chemokines, which induce cell chemotaxis) in ancomposition are released from the composition and induce stem cells tomigrate towards the amnion composition. In one aspect, the recruitmentmay be indirect, wherein stem cell recruiting factors in the compositionare released from the composition which induce nearby cells to releasefactors (e.g. chemokines), that in turn induce stem cells to migratetowards the composition. Still further, stem cell recruitment may embodyboth direct and indirect factors.

As used herein “cancer amenable to treatment with a metallic ion” referto those cancers that are treated with one or more metallic ions, e.g.platinum alkylators, non-limiting examples of which include cisplatin,carboplatin, oxaliplatin, satraplatin, as are well known to the skilledartisan. Non-limiting examples of such cancers include non-small celllung, breast, ovarian, testicular, prostate, and bladder cancer.

“Alkylators” or “alkylating agents” as used herein are agents that canalkylate, or transfer an electrophilic metal moiety, such as a platinummoiety to, nucleic acids and/or proteins as is well known to the skilledartisan. Alkylating agents may also be alkylating-like agents, whichrefers to agents that do not contain alkyl groups but have a mode ofaction similar to alkylating agents.

“Pharmacologically active agent comprising metal ions” refers to asubstance containing an active mental ion, wherein the therapeuticeffect of the agent, such as treatment of a disease, injury orcondition, is at least in part due to the activity of the mental ion.Pharmacologically active agent comprising metal ions include platinumalkylating agents. “Pharmacologically active metal ion” refers to theactive metal ion the pharmacologically active agent comprising metalions, such as platinum ion, silver ion, copper ion, etc.Pharmacologically active metal ions are known in the art asantimicrobial agents and anticancer agents. U.S. Patent ApplicationPublication Nos. US2014/0141096, US2014/0142041, and U.S. patentapplication Ser. Nos. 13/903,878, and 13/860,473 describepharmacologically active metal ions and their uses. These references areincorporated herein by reference in their entirety.

The term “diseased” as used herein refers to an organ and/or body partthat is characterized as being in a disease state, or susceptible tobeing in a disease state, wherein the disease is amenable to treatmentwith stem cells.

The term “injured” as used herein is used to have an ordinary meaning inthe art, and includes any and all types of damage to an organ and/orbody part, wherein the injury is amenable to treatment with stem cells.

The term “implantable” and derivatives thereof means the device can beinserted, embedded, grafted or otherwise acutely or chronically attachedor placed in or on a subject.

Titles or subtitles may be used in the specification for the convenienceof a reader, which are not intended to influence the scope of thepresent invention. Additionally, some terms used in this specificationare more specifically defined below.

The term “treatment” or “treating”, to the extent it relates to adisease or condition, includes preventing the disease or condition fromoccurring or reoccurring, inhibiting development of the disease orcondition, reducing or eliminating the disease or condition, and/orrelieving one or more symptoms of the disease or condition.

I. Compositions Comprising Micronized Placental and Chelators

Described herein are micronized placental composition, such as amnion orchorion compositions and/or filler with one or more chelating moietiesbound thereto and pharmaceutical compositions thereof. Such compositionsare prepared from micronized placental tissue components, which aredescribed in International Patent Application WO2012/112410, as well asin U.S. provisional application Ser. Nos. 61/442,346, 61/543,995, andUS2014/0050788. The contents of these applications are specificallyincorporated by reference in their entireties. It is understood that theterm “micronized” is meant to include micron and sub-micron sizedplacental tissue particles.

In one aspect, the invention is directed to a composition that includes(a) micronized amnion and optionally at least one of micronized chorion,intermediate tissue layer, or any combination thereof, (b) one or morechelating moieties, wherein the chelating moieties are covalently boundto the amnion, and optionally (c) a pharmaceutically acceptableexcipient.

In one aspect, the invention is directed to a composition that includes(a) micronized chorion and optionally at least one of micronized amnion,intermediate tissue layer, or any combination thereof, (b) one or morechelating moieties, wherein the chelating moieties are covalently boundto the chorion, and optionally (c) a pharmaceutically acceptableexcipient.

In one aspect, the invention is directed to a composition that includes(a) micronized laminated layers of placental tissues comprising twoamnion layers, two chorion layers, or one amnion layer and one chorionlayers, and optionally at least one of micronized amnion, intermediatetissue layer, or any combination thereof, (b) one or more chelatingmoieties, wherein the chelating moieties are covalently bound to theamnion and/or chorion, and optionally (c) a pharmaceutically acceptableexcipient.

In another aspect, the invention is directed to a composition thatincludes (a) micronized amnion and optionally at least one of micronizedchorion, intermediate tissue layer, or any combination thereof, (b) oneor more fillers, and (c) one or more chelating moieties, wherein thechelating moieties are covalently bound to the amnion and/or the filler,and optionally (d) a pharmaceutically acceptable excipient.

In another aspect, the invention is directed to a composition thatincludes (a) micronized chorion and optionally at least one ofmicronized amnion, intermediate tissue layer, or any combinationthereof, (b) one or more fillers, and (c) one or more chelatingmoieties, wherein the chelating moieties are covalently bound to thechorion and/or the filler, and optionally (d) a pharmaceuticallyacceptable excipient.

For example, the composition includes micronized amnion with one or morechelating moieties bound thereto, which can be used without the additionof any fillers, stabilizers, buffers, or pharmaceutical components.Alternatively, the composition includes micronized placental tissue andat least one pharmaceutical excipient such as a filler, a stabilizer, abuffer, a coloring agent, a disintegrating agent and the like andoptionally one or more pharmaceutical components. Preferably, thecoloring agent facilitates in locating and properly placing theplacental tissue, which can be otherwise difficult to discern fromnormal tissue, to the intended treatment site. The disintegrating agentmodifies the rate that the micronized placental tissue compositionerodes or disintegrates in vivo after being introduced to a subject.

In an embodiment, an effective amount of a pharmacologically activemetal ion is chelated to a component of the composition. In anembodiment, the pharmacologically active metal ion is an anticanceragent. In an embodiment, the pharmacologically active metal ion isplatinum ion.

In an embodiment, the micronized placental composition, such as amnionor chorion composition is optionally molded and dehydrated. In oneembodiment, the molded composition further comprises a filler. One ormore chelating moieties may be bound to the amnion, filler, or both. Asone of ordinary skill in the art would understand, the molded,dehydrated composition is compressed under pressure into any shape orsize, as long as the molded placental tissue composition has a coherentmass having a certain density. Alternatively, the dehydrated micronizedplacental components are compressed into any mold having a desired shapeor size such that the molded dehydrated placental tissue graft takes theshape and size of the mold. It is within the purview of one of ordinaryskill in the art to select suitable molding material, such as silicone,resin, Teflon®, or stainless steel, to form a mold of desired shape andsize. Examples of molded placental tissue and methods of making can befound in U.S. patent application Ser. No. 13/815,753, which is herebyincorporated by reference in its entirety.

The molded composition optionally further comprises a biologicallycompatible plasticizer. The type and amount of plasticizer can bedetermined based on the desired properties of the molded composition,for example strength, flexibility, hydrophobicity, or hydrophilicity.

II. Methods of Making Placental Compositions with One or More ChelatingMoieties Bound Thereto

FIGS. 1A and 1B depict an overview (100) and certain aspects of thesteps to harvest, process, and prepare dehydrated micronized placentalmaterial. More detailed descriptions and discussion regarding eachindividual step will follow. Initially, the placental tissue iscollected from a consenting patient following an elective Cesareansurgery (step 110). The material is preserved and transported inconventional tissue preservation manner to a suitable processinglocation or facility for check-in and evaluation (step 120). Grossprocessing, handling, and separation of the tissue layers then takesplace (step 130). Acceptable tissue is then decontaminated (step 140)and dehydrated (step 145). After decontamination and dehydration, theplacental tissue components (e.g., amnion, intermediate tissue layerand/or chorion individually or as grafts) are then micronized (step150). Chelators can be bound to the placental component (step 170)before (FIG. 1B) or after (FIG. 1A) dehydration or after micronization.Chelators may be bound to the filler at any step. Conjugation of thechelators to the placental component may optionally be performed aftermicronization. The micronized placental component and any othercomponents are optionally compressed/molded under pressure into adesired shape or size. Each step is described in detail below.

Initial Tissue Collection (Step 110)

The components used to produce the placental composition describedherein are derived from the placenta. The source of the placenta canvary. In one aspect, the placenta is derived from a mammal such as humanand other animals including, but not limited to, cows, pigs, and thelike can be used herein. In the case of humans, the recovery of theplacenta originates in a hospital, where it is preferably collectedduring a Cesarean section birth. The donor, referring to the mother whois about to give birth, voluntarily submits to a comprehensive screeningprocess designed to provide the safest tissue possible fortransplantation. The screening process preferably tests for antibodiesto the human immunodeficiency virus type 1 and type 2 (anti-HIV-1 andanti-HIV-2), antibodies to the hepatitis B virus (anti-HBV) hepatitis Bsurface antigens (HBsAg), antibodies to the hepatitis C virus(anti-HCV), antibodies to the human T-lymphotropic virus type I and typeII (anti-HTLV-I, anti-HTLV-II), CMV, and syphilis, and nucleic acidtesting for human immune-deficiency virus type 1 (HIV-1) and for thehepatitis C virus (HCV), using conventional serological tests. The abovelist of tests is exemplary only, as more, fewer, or different tests maybe desired or necessary over time or based upon the intended use of thegrafts, as will be appreciated by those skilled in the art.

Based upon a review of the donor's information and screening testresults, the donor will either be deemed acceptable or not. In addition,at the time of delivery, cultures are taken to determine the presence ofbacteria, for example, Clostridium or Streptococcus. If the donor'sinformation, screening tests, and the delivery cultures are allsatisfactory (i.e., do not indicate any risks or indicate acceptablelevel of risk), the donor is approved by a medical director and thetissue specimen is designated as initially eligible for furtherprocessing and evaluation.

Human placentas that meet the above selection criteria are preferablybagged in a saline solution in a sterile shipment bag and stored in acontainer of wet ice for shipment to a processing location or laboratoryfor further processing.

If the placenta is collected prior to the completion of obtaining theresults from the screening tests and delivery cultures, such tissue islabeled and kept in quarantine. The placenta is approved for furtherprocessing only after the required screening assessments and deliverycultures, which declare the tissue safe for handling and use, aresatisfied and final approval is obtained from a medical director.

Material Check-in and Evaluation (Step 120)

Upon arrival at the processing center or laboratory, the shipment isopened and verified that the sterile shipment bag/container is stillsealed and in the coolant, that the appropriate donor paperwork ispresent, and that the donor number on the paperwork matches the numberon the sterile shipment bag containing the tissue. The sterile shipmentbag containing the tissue is then stored in a refrigerator until readyfor further processing.

Gross Tissue Processing (Step 130)

When the tissue is ready to be processed further, the sterile suppliesnecessary for processing the placental tissue further are assembled in astaging area in a controlled environment and are prepared forintroduction into a controlled environment. In one aspect, the placentais processed at room temperature. If the controlled environment is amanufacturing hood, the sterile supplies are opened and placed into thehood using conventional sterilization techniques. If the controlledenvironment is a clean room, the sterile supplies are opened and placedon a cart covered by a sterile drape. All the work surfaces are coveredby a piece of sterile drape using conventional sterilization techniques,and the sterile supplies and the processing equipment are placed ontothe sterile drape, again using conventional sterilization techniques.

Processing equipment is decontaminated according to conventional andindustry-approved decontamination procedures and then introduced intothe controlled environment. The equipment is strategically placed withinthe controlled environment to minimize the chance for the equipment tocome in proximity to or be inadvertently contaminated by the tissuespecimen.

Next, the placenta is removed from the sterile shipment bag andtransferred aseptically to a sterile processing basin within thecontrolled environment. The sterile basin contains hypertonic salinesolution (e.g., 18% NaCl) that is at or near room temperature. Theplacenta is gently massaged to help separate blood clots and to allowthe placental tissue to reach room temperature, which facilitates theseparation of the placental components from each other (e.g., amnionmembrane and chorion). After having warmed up to ambient temperature(e.g., after about 10-30 minutes), the placenta is then removed from thesterile processing basin and laid flat on a processing tray with theamnion membrane layer facing down for inspection.

The placenta is examined for discoloration, debris or othercontamination, odor, and signs of damage. The size of the tissue is alsonoted. A determination is made, at this point, as to whether the tissueis acceptable for further processing.

The amnion and chorion are next carefully separated. In one aspect, thematerials and equipment used in this procedure include a processingtray, 18% saline solution, sterile 4×4 sponges, and two sterile Nalgenejars. The placenta tissue is then closely examined to find an area(typically a corner) in which the amnion can be separated from thechorion. The amnion appears as a thin, opaque layer on the chorion.

The fibroblast layer is identified by gently contacting each side of theamnion with a piece of sterile gauze or a cotton tipped applicator. Thefibroblast layer will stick to the test material. The amnion is placedinto processing tray basement membrane layer down. Using a bluntinstrument, a cell scraper, or sterile gauze, any residual blood is alsoremoved. This step must be done with adequate care, again, so as not totear the amnion. The cleaning of the amnion is complete once the amnionis smooth and opaque-white in appearance.

In certain aspects, the intermediate tissue layer, also referred to asthe spongy layer, is substantially removed from the amnion in order toexpose the fibroblast layer. The term “substantially removed” withrespect to the amount of intermediate tissue layer removed is definedherein as removing greater than 90%, greater than 95%, or greater than99% of the intermediate tissue layer from the amnion. This can beperformed by peeling the intermediate tissue layer from the amnion.Alternatively, the intermediate tissue layer can be removed from theamnion by wiping the intermediate tissue layer with gauze or othersuitable wipe. The resulting amnion can be subsequently decontaminatedusing the process described below.

In certain aspects, the epithelium layer present on the amnion issubstantially removed in order to expose the basement layer of theamnion. The term “substantially removed” with respect to the amount ofepithelium removed is defined herein as removing greater than 90%,greater than 95%, or greater than 99% of the epithelial cells from theamnion. The presence or absence of epithelial cells remaining on theamnion layer can be evaluated using techniques known in the art. Forexample, after removal of the epithelial cell layer, a representativetissue sample from the processing lot is placed onto a standardmicroscope examination slide. The tissue sample is then stained usingEosin Y Stain and evaluated as described below. The sample is thencovered and allowed to stand. Once an adequate amount of time has passedto allow for staining, visual observation is done under magnification.

The epithelium layer can be removed by techniques known in the art. Forexample, the epithelium layer can be scraped off of the amnion using acell scraper. Other techniques include, but are not limited to, freezingthe membrane, physical removal using a cell scraper, or exposing theepithelial cells to nonionic detergents, anionic detergents, andnucleases. The de-epithelialized tissue is then evaluated to determinethat the basement membrane has not been compromised and remains intact.This step is performed after completion of the processing step andbefore the tissue has been dehydrated as described in the next section.For example, a representative sample graft is removed for microscopicanalysis. The tissue sample is placed onto a standard slide, stainedwith Eosin Y and viewed under the microscope. If epithelium is present,it will appear as cobblestone-shaped cells.

The methods described herein do not remove all cellular components inthe amnion. This technique is referred to in the art as“decellularization.” Decellularization generally involves the physicaland/or chemical removal of all cells present in the amnion, whichincludes epithelial cells and fibroblast cells. For example, althoughthe removal of epithelial cells is optional, the fibroblast layerpresent in the amnion stromal layer is intact, even if the intermediatetissue layer is removed. Here, fibroblast cells are present in thefibroblast layer.

When the placental tissue is Wharton's jelly, the following exemplaryprocedure can be used. Using a scalpel or scissors, the umbilical cordis dissected away from the chorionic disk. Once the veins and the arteryhave been identified, the cord is dissected lengthwise down one of theveins or the artery. Once the umbilical cord has been dissected,surgical scissors and forceps can be used to dissect the vein and arterywalls from the Wharton's jelly. Next, the outer layer of amnion isremoved from the Wharton's jelly by cutting the amnion. Here, the outermembrane of the umbilical cord is removed such that Wharton's jelly isthe only remaining component. Thus, the Wharton's jelly as used hereindoes not include the outer umbilical cord membrane and umbilical cordvessels. The Wharton's jelly can be cut into strips. In one aspect, thestrips are approximately 1-4 cm by 10-30 cm with an approximatethickness of 1.25 cm; however, other thicknesses are possible dependingon the application.

Chemical Decontamination (Step 140)

The placental tissue can be chemically decontaminated using thetechniques described below. In one aspect, the amnion is decontaminatedat room temperature. In one aspect, the amnion produced in step 130(e.g., with or without the intermediate tissue layer) can be placed intoa sterile Nalgene jar for the next step. In one aspect, the followingprocedure can be used to clean the amnion. A Nalgene jar is asepticallyfilled with 18% saline hypertonic solution and sealed (or sealed with atop). The jar is then placed on a rocker platform and agitated forbetween 30 and 90 minutes, which further cleans the amnion ofcontaminants. If the rocker platform was not in the critical environment(e.g., the manufacturing hood), the Nalgene jar is returned to thecontrolled/sterile environment and opened. Using sterile forceps or byaseptically decanting the contents, the amnion is gently removed fromthe Nalgene jar containing the 18% hypertonic saline solution and placedinto an empty Nalgene jar. This empty Nalgene jar with the amnion isthen aseptically filled with a pre-mixed antibiotic solution. In oneaspect, the premixed antibiotic solution is composed of a cocktail ofantibiotics, such as Streptomycin Sulfate and Gentamicin Sulfate. Otherantibiotics, such as Polymixin B Sulfate and Bacitracin, or similarantibiotics now available or available in the future, are also suitable.Additionally, it is preferred that the antibiotic solution be at roomtemperature when added so that it does not change the temperature of orotherwise damage the amnion. This jar or container containing the amnionand antibiotics is then sealed or closed and placed on a rocker platformand agitated for, preferably, between 60 and 90 minutes. Such rocking oragitation of the amnion within the antibiotic solution further cleansthe tissue of contaminants and bacteria. Optionally, the amnion can bewashed with a detergent. In one aspect, the amnion can be washed with0.1 to 10%, 0.1 to 5%, 0.1 to 1%, or 0.5% Triton-X wash solution.

If the rocker platform was not in the critical environment (e.g., themanufacturing hood), the jar or container containing the amnion andantibiotics is then returned to the critical/sterile environment andopened. Using sterile forceps, the amnion is gently removed from the jaror container and placed in a sterile basin containing sterile water ornormal saline (0.9% saline solution). The amnion is allowed to soak inplace in the sterile water/normal saline solution for at least 10 to 15minutes. The amnion may be slightly agitated to facilitate removal ofthe antibiotic solution and any other contaminants from the tissue.After at least 10 to 15 minutes, the amnion is ready to be dehydratedand processed further.

In the case of chorion, the following exemplary procedure can be used.After separation of the chorion from the amnion and removal of clottedblood from the fibrous layer, the chorion is rinsed in 18% salinesolution for 15 minutes to 60 minutes. During the first rinse cycle, 18%saline is heated in a sterile container using a laboratory heating platesuch that the solution temperature is approximately 48° C. The solutionis decanted, the chorion tissue is placed into the sterile container,and decanted saline solution is poured into the container. The containeris sealed and placed on a rocker plate and agitated for 15 minutes to 60minutes. After 1 hour agitation bath, the chorion tissue was removed andplaced into second heated agitation bath for an additional 15 minutes to60 minutes rinse cycle. Optionally, the chorion tissue can be washedwith a detergent (e.g., Triton-X wash solution) as discussed above forthe decontamination of amnion. The container is sealed and agitatedwithout heat for 15 minutes to 120 minutes. The chorion tissue is nextwashed with deionized water (250 ml of DI water×4) with vigorous motionfor each rinse. The tissue is removed and placed into a container of1×PBS w/EDTA solution. The container is sealed and agitated for 1 hourat controlled temperature for 8 hours. The chorion tissue is removed andrinsed using sterile water. A visual inspection was performed to removeany remaining discolored fibrous blood material from the chorion tissue.The chorion tissue should have a cream white visual appearance with noevidence of brownish discoloration.

The following exemplary procedure can be used when the placental tissueis Wharton's jelly. The Wharton's jelly is transferred to a sterileNalgene jar. Next, room temperature 18% hypertonic saline solution isadded to rinse the tissue and the jar is sealed. The jar is agitated for30 to 60 minutes. After incubation, the jar is decontaminated andreturned to the sterile field. The tissue is transferred to a cleansterile Nalgene jar and prewarmed (about 48° C.) with 18% NaCl. Thecontainer is sealed and placed on rocker plate and agitated for 60 to 90minutes.

After the rinse, the jar is decontaminated and returned to the sterilefield. The tissue is removed and placed into an antibiotic solution. Thecontainer is sealed and agitated for 60 to 90 minutes on a rockerplatform. Following incubation, the jar may be refrigerated at 1° C. to10° C. for up to 24 hours.

The Wharton's jelly is next transferred to a sterile basin containingapproximately 200 mL of sterile water. The tissue is rinsed for 1-2minutes and transferred to a sterile Nalgene jar containingapproximately 300 ml of sterile water. The jar is sealed and placed onthe rocker for 30 to 60 minutes. After incubation, the jar is returnedto the sterile field. The Wharton's jelly should have a cream whitevisual appearance with no evidence of brownish discoloration.

Dehydration (Step 145)

In one aspect, the placental tissue or components thereof as describedabove, or any combination thereof, can be processed into tissue grafts(i.e., laminates) that are subsequently micronized. In another aspect,the placental tissue or individual components thereof can be dehydratedindependently and subsequently micronized alone or as a mixture ofcomponents. In one aspect, the tissue (i.e., individual membrane orgraft) is dehydrated by chemical dehydration followed by freeze-drying.In one aspect, the chemical dehydration step is performed by contactingthe amnion, chorion, and/or intermediate layer with a polar organicsolvent for a sufficient time and amount in order to substantially(i.e., greater than 90%, greater than 95%, or greater than 99%) orcompletely remove residual water present in the tissue (i.e., dehydratethe tissue). The solvent can be protic or aprotic. Examples of polarorganic solvents useful herein include, but are not limited to,alcohols, ketones, ethers, aldehydes, or any combination thereof.Specific, non-limiting examples include DMSO, acetone, tetrahydrofuran,ethanol, isopropanol, or any combination thereof. In one aspect, theplacental tissue is contacted with a polar organic solvent at roomtemperature. No additional steps are required, and the tissue can befreeze-dried directly as discussed below.

After chemical dehydration, the tissue is freeze-dried in order toremove any residual water and polar organic solvent. In one aspect, theamnion, chorion, and/or intermediate layer can be laid on a suitabledrying fixture prior to freeze-drying. For example, one or more stripsof amnion can be laid on a suitable drying fixture. Next, chorion islaid on top of the amnion. In this aspect, an amnion/chorion tissuegraft is produced. Alternatively, a strip of amnion can be placed on afirst drying fixture, and a strip of chorion can be placed on a seconddrying fixture. The drying fixture is preferably sized to be largeenough to receive the placental tissue, fully, in laid out, flatfashion. In one aspect, the drying fixture is made of Teflon® or ofDelrin®, which is the brand name for an acetal resin engineering plasticinvented and sold by DuPont and which is also available commerciallyfrom Werner Machine, Inc. in Marietta, Ga. Any other suitable materialthat is heat and cut resistant, capable of being formed into anappropriate shape to receive wet tissue can also be used for the dryingfixture.

Once the tissue is placed on the drying fixture, the drying fixture isplaced in the freeze-dryer. The use of the freeze-dryer to dehydrate thetissue can be more efficient and thorough compared to other techniquessuch as thermal dehydration. In general, it is desirable to avoid icecrystal formation in the placental tissue as this may damage theextracellular matrix in the tissue. By chemically dehydrating theplacental tissue prior to freeze-drying, this problem can be avoided.

In another aspect, the dehydration step involves applying heat to thetissue. In one aspect, the amnion, chorion, and/or intermediate layer islaid on a suitable drying fixture (either as individual strips or as alaminate discussed above), and the drying fixture is placed in a sterileTyvek® (or similar, breathable, heat-resistant, and sealable material)dehydration bag and sealed. The breathable dehydration bag prevents thetissue from drying too quickly. If multiple drying fixtures are beingprocessed simultaneously, each drying fixture is either placed in itsown Tyvek® bag or, alternatively, placed into a suitable mounting framethat is designed to hold multiple drying frames thereon and the entireframe is then placed into a larger, single sterile Tyvek® dehydrationbag and sealed.

The Tyvek® dehydration bag containing the one or more drying fixtures isthen placed into a non-vacuum oven or incubator that has been preheatedto approximately 35 to 50 degrees Celsius. The Tyvek® bag remains in theoven for between 30 to 120 minutes. In one aspect, the heating step canbe performed at 45 minutes at a temperature of approximately 45° C. todry the tissue sufficiently but without over-drying or burning thetissue. The specific temperature and time for any specific oven willneed to be calibrated and adjusted based on other factors includingaltitude, size of the oven, accuracy of the oven temperature, materialused for the drying fixture, number of drying fixtures being driedsimultaneously, whether a single or multiple frames of drying fixturesare dried simultaneously, and the like.

In one aspect, the placental tissue can be dehydrated using adehydration device which enhances the rate and uniformity of thedehydration process. Representative dehydration device suitable fordrying placental tissue grafts are described in U.S. Patent publicationNo. 2014/0051059. The contents of this application is incorporated byreference in their entireties.

Preparation of Micronized Placental Components (Step 150)

Once the placental tissue or components thereof as described above havebeen dehydrated individually or in the form of tissue graft, thedehydrated tissue(s) is micronized. The micronized placental componentscan be produced using instruments known in the art. For example, theRetsch Oscillating Mill MM400 can be used to produce the micronizedcompositions described herein. The particle size of the materials in themicronized composition can vary as well depending upon the applicationof the micronized composition. In one aspect, the micronized compositionhas particles that are less than 500 μm, less than 400 μm, less than 300μm, less than 200 μm, less than 100 μm, less than 50 μm, less than 25μm, less than 20 μm, less than 15 μm, less than 10 μm, less than 9 μm,less than 8 μm, less than 7 μm, less than 6 μm, less than 5 μm, lessthan 4 μm, less than 3 μm, less than 2 μm, or from 2 μm to 400 μm, from25 μm to 300 μm, from 25 μm to 200 μm, or from 25 μm to 150 μm. In oneaspect, the micronized composition has particles that have a diameterless than 150 μm, less than 100 μm, or less than 50 μm. In otheraspects, particles having a larger diameter (e.g. 150 μm to 350 μm) aredesirable. In all cases, the diameter of the particle is measured alongits longest axis.

In one embodiment, the size of the particles may be reduced tonano-range. As one skilled in the art would understand, nanoparticles ofplacental components may be desirable for the increased density and/orincreased release rate upon applying to the wound. Preferably, theparticle size of the micronized particles is from about 0.05 μm to about2 μm, from about 0.1 μm to about 1.0 μm, from about 0.2 μm to about 0.8μm, from about 0.3 μm to about 0.7 μm, or from about 0.4 μm to about 0.6μm. Alternatively, the particle size of the micronized particles is atleast 0.05 μm, at least 0.1 μm, at least 0.2 μm, at least 0.3 μm, atleast 0.4 μm, at least 0.5 μm, at least 0.6 μm, at least 0.7 μm, atleast 0.8 μm, at least 0.9 μm, or at least 1 μm. Alternatively, theparticle size of the micronized particles is less than 1 μm, less than0.9 μm, less than 0.8 μm, less than 0.7 μm, less than 0.6 μm, less than0.5 μm, less than 0.4 μm, less than 0.3 μm, less than 0.2 μm, less than0.1 μm, or less than 0.05 μm.

In one aspect, the initial micronization is performed by mechanicalgrinding or shredding. In another aspect, micronization is performed bycryogenic grinding. In this aspect, the grinding jar containing thetissue is continually cooled with liquid nitrogen from the integratedcooling system before and during the grinding process. Thus, the sampleis embrittled and volatile components are preserved. Moreover, thedenaturing of proteins in the amnion, intermediate tissue layer, and/orchorion is minimized or prevented. In one aspect, the CryoMillmanufactured by Retsch can be used in this aspect.

The selection of components used to make the placental composition, suchas amnion or chorion composition described herein can vary dependingupon the end-use of the placental tissue composition For example,placental tissue or individual components such as amnion, chorion,intermediate tissue layer, Wharton's jelly or any combination thereofcan be admixed with one another and subsequently micronized. In anotheraspect, one or more tissue grafts composed of one or more placentaltissue, amnion, chorion, intermediate tissue layers, or any combinationthereof (i.e., laminates) can be micronized. In a further aspect, one ormore tissue grafts composed of one or more amnion, chorion, intermediatetissue layers, or any combination can be admixed with amnion, chorion,intermediate tissue layer, or any combination thereof as individualcomponents and subsequently micronized.

The amount of different components can vary depending upon theapplication of the amnion composition. In one aspect, the amnioncomposition is composed solely of amnion (with or without theintermediate tissue layer). In one aspect, when the amnion compositionis composed of amnion (with or without the intermediate tissue layer)and intermediate tissue layer, the weight ratio of amnion tointermediate tissue layer is from 10:1 to 1:10, 9:1 to 1:1, 8:1 to 1:1,7:1 to 1:1, 6:1 to 1:1, 5:1 to 1:1, 4:1 to 1:1, 3:1 to 1:1, 2:1 to 1:1,or about 1:1. In another aspect, when the amnion composition is composedof amnion (with or without the intermediate tissue layer) and chorion,the weight ratio of chorion to amnion is from 10:1 to 1:10, 9:1 to 1:1,8:1 to 1:1, 7:1 to 1:1, 6:1 to 1:1, 5:1 to 1:1, 4:1 to 1:1, 3:1 to 1:1,2:1 to 1:1, or about 1:1.

Separation of particle sizes can be achieved by fractionation of themicronized material in sterile water by forming a suspension ofparticles. The upper most portion of the suspension will containpredominantly the smallest particles and the lower most portion of thesuspension will contain predominantly the heaviest particles.Alternatively, the micronized material can be fractionated using sievesof the desired size(s). Fractionation leads to particle size separationand repeated fractionation will lead to separation of the micronizedparticles into varying sizes. The so separated particles can berecombined in the desired ratio of particle size as is most appropriatefor making the amnion composition and the desired medical application.

Fillers

In some aspects, the compositions of the present invention comprise oneor more fillers. In some aspects, the fillers are biocompatiblepolymers. Suitable polymers include those that are known in the art andare capable of forming chelator conjugates as described herein.Preferred biocompatible polymers useful in this invention includebiodegradable polymers.

Suitable polymers include, without limitation, naturally-occurringpolymers, synthetic polymers or mixtures thereof. In one embodiment, thepolymer includes collagen (such as human collagen) or collagen preparedfrom placental tissue, e.g., amnion or chorion containing collagen.Other examples of naturally-occurring biocompatible polymers include,but are not limited to, hylauronic acid, fibrin, fibrous or globularproteins, complex carbohydrates, glycosaminoglycans, such as, ormixtures thereof. Thus, in one embodiment, the polymer may includecollagens of all types, elastin, laminin, hyaluronic acid, alginic acid,desmin, versican, fibrin, fibronectin, vitronectin, albumin, and thelike. Exemplary synthetic biocompatible polymers include, but are notlimited to, polyoxyalkylenes (e.g., polyoxyethylene, polyoxypropylene,copolymers of oxyethylene and oxypropylene, and the like), polyethyleneglycol, polymethylene glycol, polytrimethylene glycols,polyvinylpyrrolidones, caprolactones, 2-hydroxyethyl methacrylate(HEMA), silicone such as Nusil MED-6215 or other silicone suitable forimplantation, poly(epsilon-caprolactone) dimethylacrylate, polysulfone,(poly)methyl methacrylate (PMMA), soluble Teflon-AF, poly ethyleneteraphthalate (PET, Dacron), Nylon, polyvinyl alcohol, polyurethane,hydroxyapatite, and the like and mixtures thereof. Such polymerspreferably have an average molecular weight of at least about 10,000 andmore preferably from about 10,000 to about 1,000,000. In someembodiments, these polymers preferably have an average molecular weightof at least 10,000 and more preferably from about 10,000 to about100,000. The polymers described herein can be either cross-linked withnon-chelating agents or non-cross-linked. Common non-chelatingcross-linking agents include carbodimides, diisothiocyanates,dicarboxylic acids, diamines and the like.

In some embodiments, the biocompatible polymers are water solublepolymers and are sometimes referred to as hydrophilic polymers. Watersolubility can be achieved incorporating a sufficient number of oxygen(or less frequently nitrogen) atoms available for forming hydrogen bondsin aqueous solution. Hydrophilic polymers include, without limitation,polyoxyethylene, polyethylene glycol, polymethylene glycol,polytrimethylene glycols, polyvinylpyrrolidones, or derivatives thereof.The polymers are preferably linear or only slightly branched (i.e.,having only about 2-10 significant free ends), and will not besubstantially cross-linked. Other suitable polymers includepolyoxyethylene-polyoxypropylene block polymers and copolymers.

Polyoxyethylene-polyoxypropylene block polymers having an ethylenediamine nucleus (and thus having four ends) are also available and maybe used in the practice of the invention. Hydrophilic polymers can alsoinclude naturally occurring polymers such as proteins (e.g., and withoutlimitation, a collagen), starch, cellulose, and the like.

All suitable polymers are biocompatible, and preferably non-toxic andnon-inflammatory when administered in vivo, and will more preferably bedegradable in vivo with a degradation time of at least several months.

In some embodiments, the polymers have at least one and preferably up to1000 reactive functionalities which are complementary to the reactionfunctionalities on the precursor chelator compound. Typically, thecomplementary reactive functionality is present on the polymer such asreactive functionalities found in collagen, hylauronic acid, and thelike or can be introduced onto the polymer by conventional chemicalsynthetic techniques well known to the skilled artisan. Exemplaryfunctionalities include, without limitation, amine, carboxylic acid,hydrazine, hydrazone, azide, isocyanate, isothiocyanate, alkoxyamine,aldehyde, epoxy, nitrile, maleimide, halo, hydroxyl, thiol or acombination thereof. Preferably, the reactive functional group isselected from the amine or carboxylic acid. Complementaryfunctionalities include those that react with each other to form acovalent bond. Examples include isocyanates with amines and hydroxylgroups to form a urea or carbamate linkage, carboxylic acids and amineswhich form amides, and the like. The following table illustrates somecommon complementary reactive groups, one of which is found on theprecursor chelating moiety (first reactive functionality) and the otheron the polymer (second reactive functionality).

First reactive Second reactive Covalent functionality functionality Bondformed Amine Carboxyl Amide Hydroxyl Halide Ether Isocyanate Amine UreaIsocyanate Hydroxyl Carbamate Carboxyl Amine Amide ThioisocyanateHydroxyl Thiocarbamate

The polymers can be functionalized, for example, by introducing anamine-functional monomer, either pendant or terminal to the polymer. Asuitable method for imparting a pendant amine functionality to thepolymer is to use a monomer containing a pendant amine functionality.Suitable monomers containing a pendant amine functionality include2-aminoethylacrylate, 2-aminoethylmethacrylate, 2-aminoethylacrylamide,2-amino ethylmethacrylamide, dimethylaminoethylmethacryl, aminopropyl(meth)acrylamide and the like. When a monomer containing a pendant aminefunctionality is used, the resulting polymer may contain one or morependant amine groups. Preferably, the pendant amine functionality whichis imparted to the polymer is a terminal pendant amine functionality.Terminal pendant amine functionality can be imparted to a polymer byusing one or more compounds which, when they function as a chaintransfer agent, have a pendant amine group. Preferred compounds forimparting terminal amine functionality are amine-thiols, e.g.,N-butylaminoethanethiol, N,N-diethylaminoethanethiol and salts thereof.

Likewise, the polymers of the invention can be functionalized, forexample, by introducing a carboxylic acid-functional monomer. Preferredacid monomers are carboxylic acids or their derivatives, including, butnot limited to, monounsaturated monocarboxylic acid, monounsaturateddicarboxylic acid, anhydrides or alcohol derived mono- or di-esters.Upon reaction with the polymer, e.g., a polyolefin, the monounsaturationof the monounsaturated carboxylic reactant becomes saturated. Exemplarymonounsaturated carboxylic reactants include fumaric acid, itaconicacid, maleic acid, maleic anhydride, chloromaleic acid, chloromaleicanhydride, acrylic acid, methacrylic acid, crotonic acid, cinnamic acid,and lower alkyl acid esters, such as methyl maleate, ethyl fumarate, andmethyl fumarate. In other embodiments, polymer containing anhydride orester functionality can be converted by well known hydrolysis methods toacid.

Chelators

A variety of chelators well known for binding various pharmacologicallyactive metal ions, such as ions of copper, silver, and platinum, areuseful in the present invention; preferably such chelators arebiocompatible. Biocompatible chelators are described, for example, inU.S. Patent Application Ser. No. 61/728,198 and U.S. Patent ApplicationPublication No. 2014/0142041, both of which are incorporated herein byreference in their entireties.

Suitable chelators preferably comprise one or more C₂-C₁₀ alkyl orheteroalkyl, C₆-C₁₀ aryl C₃-C₁₀ heteroaryl, or C₅-C₁₀ cycloalkyl groupssubstituted with at least two, adjacent (or 1,2 substituted) hydroxy,C₂-C₁₀ alkoxy, amino, (C₂-C₁₀) alkylamino, (C₂-C₁₀)₂dialkylamino,mercapto, C₂-C₁₀ thioalkyl groups, which alkyl or heteroalkyl, aryl,heteroaryl, or cycloalkyl groups are attached via a linker, or a bondand a functional joining group, to the amnion and/or filler.

The term “chelator precursor compound” refers to the chelator prior toreaction with the amnion composition or filler. The precursor compoundcontains a reactive functionality which reacts with a complementaryfunctionality on the amnion or filler to form a covalent bond. Theresulting chelating moiety is referred to as the “chelator” and isdefined above. The reactive functional groups on the chelator precursorcompounds form a stable covalent bond when coupled with thecomplementary reactive functional group on the amnion or filler. Suchstable covalent bonds include by way of example esters, ethers, amides(—CONH—, —NHCO—, —N(alkyl)CO— or CO—N(alkyl)-), carbamates, urea,carbonate, thiocarbonate, thiourea, carbamate, and urethane bonds, aswell as any other well-known covalent bonds. Reactive functional groupseither on the chelator precursor compound, the amnion, or the fillerinclude those such as amino, hydroxy, mercapto, and carboxylic acid,carboxylate esters, isocyanate, and other well-known functional groupsthat can be chemically bonded following art known methods to acomplementary reactive functional group on the amnion or filler asutilized herein. The group X on the chelator precursor compound isselected from the group consisting of H or a complimentary reactivefunctional group. It is understood that when X is a complimentaryfunctional group the other functional groups on the compound may need tobe blocked or protected using conventional methods and protectinggroups.

In one embodiment, the chelating agent comprises a 1,2-benzoquinoneand/or a 1,2-dihydroxy phenyl moiety. In another embodiment, thechelating moiety is derived from a precursor compound selected from thegroup consisting of nordihydroguaiaretic acid (NDGA),3,4-dihydroxyphenylalanine, dopamine, 3,4-dihydroxybenzaldehyde, and3,4-dihydroxybenzoic acid.

In one embodiment, the chelating agent is derived from a precursorcompound which can both react with and form a covalent bond to amnionand/or filler and reversibly bind the pharmacologically active metalion. Preferably, the precursor compound is selected from the groupconsisting of:

wherein X is H or a complimentary reactive functional group, such thatwhen X is a complimentary reactive functional group, the otherfunctional groups on the molecule are protected.

In one embodiment, the precursor compound is:

where X′ is a complimentary reactive functional group, such that thehydroxyl groups on the molecule are optionally protected.

Preferable chelating agents used are derived from naturally occurringcompounds such as dopamine and L-dopa.

After reaction, the chelator is sometimes referred to a chelating agentor as a chelating moiety.

Pharmacologically Active Metal Ions

Suitable pharmacologically active metals are known in the art. In oneembodiment, the pharmacologically active metal is an anticancer agent.Anticancer metals include, without limitation, platinum, ruthenium,osmium and cobalt. As used herein, the term “anticancer agent” or“anticancer metal ion” refers to any metal ion-containing compound thatcan improve one or more cancer symptoms, and/or ameliorate one or morecancer-side effects, and/or prevent and/or impede invasiveness and/ormetastasis of cancer.

Anticancer agents are detailed in U.S. Patent Application PublicationNo. 2014/0142041, which is incorporated by reference in its entirety. Inone embodiment, the metal ion is ionic platinum. In one embodiment, theplatinum comprises platinum (II) and/or platinum (IV). In anotherembodiment, the platinum comprises a compound selected from the group of

denotes binding to the chelator.

The chelated amnion-Pt and/or filler-Pt constructs of this invention areprepared by contacting appropriate Pt salts, preferably, Pt(II) saltswith a functionalized amnion composition or filler of this invention.Preferably, the Pt salt is a cis diamino Pt(II) dichloro or, yet morepreferably, a cis diamino Pt(II) diaqua salt. An illustrative andnon-limiting example is shown below:

Step A

Step B

Excess Pt salts, not bound to the amnion composition or filler of thisinvention, can be removed by complexing with a resin, such as Chelex. Anamnion composition-Pt(II) or filler-Pt(II) construct can be converted tothe corresponding Pt(IV) construct by oxidation, for example with H₂O₂.FIGS. 2 and 3 illustrate the use of different conjugates in a mannerwhere the release rates of the conjugates overlap (FIG. 2) so as toprovide a continuous release of the metal ion whereas FIG. 3 illustratesthe use of different conjugates in a manner where the release rates donot overlap so as to provide two separate releases (bolus) of metal ion.

As used herein, a cis-diaqua platinum complex contains 2 water moleculesbound to platinum in cis or adjacent configuration, and a cis-dihaloplatinum complex contains 2 halogen, preferably chloride groups, boundto platinum in cis or adjacent configuration.

Polymer Conjugates

The polymer constructs of this invention are described below usingcollagen as an exemplary polymer. It is understood, however, thatpolymers other than collagen, such as those described herein, can beutilized in place of collagen. Exemplary biocompatible collagenconjugates and devices made therefrom are described in U.S. Pat. No.7,901,455; U.S. Patent Application Publication Nos. 2008/0161917,2008/0188933, 2008/0200992, 2009/0216233, 2009/0287308, 2010/0094318,2010/0094404, 2011/0282448, 20110282447 and 2014/0172096, which areincorporated herein by reference.

The conjugates of the present invention can be dry or partiallyhydrated. The term “dry” as used herein means the construct has amoisture content of less than about 5% by weight of the construct. Theterm “partially hydrated” as used herein means that the construct has amoisture content that is less than about 50%, typically less than about75% of the moisture content at full hydration, measured ex vivo after 24hours in a saline bath at ambient conditions. Thus, the construct canhave a moisture content of less than about 25% by weight of theconstruct, such as less than about 15% by weight of the construct.

Preparation of Molded Placental Grafts and

Pharmaceutical Compositions Thereof (Optional)

The amnion composition of the current invention may optionally be amolded placental graft. The dehydrated components, such as micronizedamnion, micronized chorion, micronized intermediate tissue layer,filler, and any combination thereof, when subjected to pressurepreferably in a non-porous mold, form a desired shape and size definedby the mold. While a porous mold is less preferred, it is contemplatedthat such can be used in the methods of this invention if water or othersolvents are allowed to escape during molding. The molded amnion grafthas a sufficient density and cohesive mass to maintain its size andshape at least until the molded amnion graft is introduced to a subject.The cohesion of the molded amnion graft is determined, in part, by theparticle size of the micronized components. For example, micronizedcomponents having larger particle size require higher compressivepressure and/or longer compression time to obtain a molded amnion grafthaving the same density as that of a molded amnion graft composed ofmicronized components having smaller particle size. In other words, formolded compositions obtained under the same compression condition, thecompositions having larger particle size have less density anddissociate at a higher rate in comparison to the compositions havingsmaller particle size.

Optionally, one or more adhesives can be admixed with the placentalcomposition, such as amnion or chorion composition prior to beingintroduced into the mold. Examples of such adhesives include, but arenot limited to, fibrin sealants, cyanoacrylates, gelatin and thrombinproducts, polyethylene glycol polymer, albumin, and glutaraldehydeproducts. The adhesives used in the process should be dehydrated priorto being mixed with the placental composition such that the mixture ofadhesives and placental composition has a sufficiently low water contentto permit compression in a non-porous mold.

In addition to the amnion, additional dehydrated components can be addedto the composition prior to and/or after micronization. In one aspect,other placental tissue components may be added. Placental tissuecomponents can comprise intermediate tissue layer, chorion, or both, aswell as dehydrated, micronized grafts of the same.

In one aspect, a dehydrated filler can be added. Examples of fillersinclude, but are not limited to, allograft pericardium, allograftacellular dermis, purified xenograft Type-1 collagen, biocellulosepolymers or copolymers, biocompatible synthetic polymer or copolymerfilms, purified small intestinal submucosa, bladder acellular matrix,cadaveric fascia, bone particles (including cancellous and cortical boneparticles), other polymers described above, or any combination thereof.A filler may be powdered, micronized, or in any other form such that thefiller can be combined with the micronized placental component.

In another aspect, a dehydrated bioactive agent can be added to thecomposition prior to and/or after micronization. Examples of bioactiveagents include, but are not limited to, naturally occurring growthfactors sourced from platelet concentrates, either using autologousblood collection and separation products, or platelet concentratessourced from expired banked blood; bone marrow aspirate; stem cellsderived from concentrated human placental cord blood stem cells;concentrated amniotic fluid stem cells or stem cells grown in abioreactor; or antibiotics. Upon application of the placentalcomposition with bioactive agent to the region of interest, thebioactive agent is delivered to the region over time. Thus, thecompositions described herein are useful as delivery devices ofbioactive agents and other pharmaceutical agents when administered to asubject. Release profiles can be modified based on, among other things,the selection of the components used to make the composition as well asthe size of the particles contained in the composition. Release profilescan further be modified by molding the composition as described above.

In some aspects, one or more stem cell recruiting factors that enhancestem cell chemotaxis and or recruitment may be added to a placentalcomposition of the present technology. In other aspects, stem cellrecruiting factors can be added to the micronized placental composition.Alternatively, stem cell recruiting factors may be added to layers of alaminate tissue graft prior to micronization. Thus, for example,cytokines, chemokines, growth factors, extracellular matrix componentsand other bioactive materials can be added to the modified amnioncomposition to enhance native stem cell recruitment. Specificnon-limiting examples of stem cell recruiting factors may include one ormore of the following: CC chemokines, CXC chemokines, C chemokines, orCX₃C chemokines. Other stem cell recruiting factors may further includegrowth factors such as α-Fibroblast Growth Factor (αFGF or αFGF-1),β-Fibroblast Growth Factor (βFGF-1 or βFGF-2), Platelet-Derived GrowthFactor (PDGF), Vascular Endothelial Growth Factor (VEGF-A, B, C, D orE), Angiopoietin-1 and -2, Insulin-like Growth Factor (IGF-1), BoneMorphogenic Protein (BMP-2 and -7), Transforming Growth Factor-α and -β(TGF-α and TGF-β), Epidermal Growth Factor (EGF), Connective TissueGrowth Factor (CTGF), Hepatocyte Growth Factor (HGF), Human GrowthHormone (HGH), Keratinocyte Growth Factor (KGF), Tumor Necrosis Factor-α(TNF-α), Leukemia Inhibitory Factor (LIF), Nerve Growth Factor (NGF),Stromal cell derived factor 1 (SDF-1α), Granulocyte Macrophage ColonyStimulating Factor (GM-CSF) and other factors as is known in the art.

Plasticizers

In yet another aspect, the placental composition is admixed with atleast one plasticizer. One skilled in the art would select a suitableplasticizer based on the biocompatibility of the plasticizer, effect ofplasticizer on the degradation or erosion rate of the placental tissuecomposition in vivo, and/or effect of the plasticizer on the strength,flexibility, consistency, hydrophobicity and/or hydrophilicity of thecomposition.

The terms “plasticizer” and “plasticizing agent” can be usedinterchangeably in the present invention. A plasticizing agent caninclude any agent or combination of agents that can be added to modifythe mechanical properties of the composition or a product formed fromthe composition.

Without intending to be bound by any theory or mechanism of action,plasticizers can be added, for example, to reduce crystallinity, lowerthe glass-transition temperature (Tg), or reduce the intermolecularforces between components within the composition, with a design goalthat may include creating or enhancing a flow between components in thecomposition. The mechanical properties that are modified include, butare not limited to, Young's modulus, tensile strength, impact strength,tear strength, and strain-to-failure. A plasticizer can be monomelic,polymeric, co-polymeric, or a combination thereof, and can be added to acomposition with or without covalent bonding. Plasticization andsolubility are analogous to the extent that selecting a plasticizerinvolves considerations similar to the considerations in selecting asolvent such as, for example, polarity. Furthermore, plasticizers canalso be added to a composition through covalent bonding that changes themolecular structure of the composition through copolymerization.

Examples of plasticizing agents include, but are not limited to, lowmolecular weight polymers such as, for example, single-block polymers,multi-block polymers, and copolymers; oligomers such as, for example,lactic acid oligomers including, but not limited to, ethyl-terminatedoligomers of lactic acid; dimers of cyclic lactic acid and glycolicacid; small organic molecules; hydrogen bond forming organic compoundswith and without hydroxyl groups; polyols such as low molecular weightpolyols having aliphatic hydroxyls; alkanols such as butanols, pentanolsand hexanols; sugar alcohols and anhydrides of sugar alcohols;polyethers such as poly(alkylene glycols); esters such as citrates,phthalates, sebacates and adipates; polyesters; aliphatic acids;saturated and unsaturated fatty acids; fatty alcohols; cholesterol;steroids; phospholipids such as, for example, lecithin; proteins such asanimal proteins and vegetable proteins; oils such as, for example, thevegetable oils and animal oils; silicones; acetylated monoglycerides;diglycerides; triglycerides; amides; acetamides; sulfoxides; sulfones;pyrrolidones; oxa acids; diglycolic acids; and any analogs, derivatives,copolymers and combinations thereof.

In some embodiments, the plasticizers include, but are not limited toother polyols such as, for example, caprolactone diol, caprolactonetriol, sorbitol, erythritol, glucidol, mannitol, sorbitol, sucrose, andtrimethylol propane. In other embodiments, the plasticizers include, butare not limited to, glycols such as, for example, ethylene glycol,diethylene glycol, Methylene glycol, tetraethylene glycol, propyleneglycol, butylene glycol, 1,2-butylene glycol, 2,3-butylene glycol,styrene glycol, pentamethylene glycol, hexamethylene glycol;glycol-ethers such as, for example, monopropylene glycol monoisopropylether, propylene glycol monoethyl ether, ethylene glycol monoethylether, and diethylene glycol monoethyl ether; and any analogs,derivatives, copolymers and combinations thereof.

In other embodiments, the plasticizers include, but are not limited toesters such as glycol esters such as, for example, diethylene glycoldibenzoate, dipropylene glycol dibenzoate, methylene glycolcaprate-caprylate; monostearates such as, for example, glycerolmonostearate; citrate esters; organic acid esters; aromatic carboxylicesters; aliphatic dicarboxylic esters; fatty acid esters such as, forexample, stearic, oleic, myristic, palmitic, and sebacic acid esters;triacetin; poly(esters) such as, for example, phthalate polyesters,adipate polyesters, glutate polyesters, phthalates such as, for example,dialkyl phthalates, dimethyl phthalate, diethyl phthalate, isopropylphthalate, dibutyl phthalate, dihexyl phthalate, dioctyl phthalate,diisononyl phthalate, and diisodecyl phthalate; sebacates such as, forexample, alkyl sebacates, dimethyl sebacate, dibutyl sebacate;hydroxyl-esters such as, for example, lactate, alkyl lactates, ethyllactate, butyl lactate, allyl glycolate, ethyl glycolate, and glycerolmonostearate; citrates such as, for example, alkyl acetyl citrates,triethyl acetyl citrate, tributyl acetyl citrate, trihexyl acetylcitrate, alkyl citrates, triethyl citrate, and tributyl citrate; estersof castor oil such as, for example, methyl ricinolate; aromaticcarboxylic esters such as, for example, trimellitic esters, benzoicesters, and terephthalic esters; aliphatic dicarboxylic esters such as,for example, dialkyl adipates, alkyl allylether diester adipates,dibutoxyethoxyethyl adipate, diisobutyl adipate, sebacic esters, azelaicesters, citric esters, and tartaric esters; and fatty acid esters suchas, for example, glycerol, mono- di- or triacetate, and sodium diethylsulfosuccinate; and any analogs, derivatives, copolymers andcombinations thereof.

In other embodiments, the plasticizers include, but are not limited toethers and polyethers such as, for example, poly(alkylene glycols) suchas poly(ethylene glycols) (PEG), polypropylene glycols), andpoly(ethylene/propylene glycols); PEG derivatives such as, for example,methoxy poly(ethylene glycol) (mPEG); and ester-ethers such as, forexample, diethylene glycol dibenzoate, dipropylene glycol dibenzoate,and triethylene glycol caprate-caprylate; and any analogs, derivatives,copolymers and combinations thereof.

In other embodiments, the plasticizers include, but are not limited to,amides such as, for example, oleic amide, erucic amide, and palmiticamide; alkyl acetamides such as, for example, dimethyl acetamide;sulfoxides such as for example, dimethyl sulfoxide; pyrrolidones suchas, for example, n-methyl pyrrolidone; sulfones such as, for example,tetramethylene sulfone; acids such as, for example, oxa monoacids, oxadiacids such as 3,6,9-trioxaundecanedioic acid, polyoxa diacids, ethylester of acetylated citric acid, butyl ester of acetylated citric acid,capryl ester of acetylated citric acid, and diglycolic acids such asdimethylol propionic acid; and any analogs, derivatives, copolymers andcombinations thereof.

In other embodiments, the plasticizers include, but are not limited tovegetable oils including, but not limited to, epoxidized soybean oil;linseed oil; castor oil; coconut oil; fractionated coconut oil;epoxidized tallates; and esters of fatty acids such as stearic, oleic,myristic, palmitic, and sebacic acid; essential oils including, but notlimited to, angelica oil, anise oil, arnica oil, aurantii aetheroleum,valerian oil, basilici aetheroleum, bergamot oil, savory oil, buccoaetheroleum, camphor, cardamomi aetheroleum, cassia oil, chenopodiumoil, chrysanthemum oil, cinae aetheroleum, citronella oil, lemon oil,citrus oil, costus oil, curcuma oil, carlina oil, elemi oil, tarragonoil, eucalyptus oil, fennel oil, pine needle oil, pine oil, filicis,aetheroleum, galbanum oil, gaultheriae aetheroleum, geranium oil, guaiacwood oil, hazelwort oil, iris oil, hypericum oil, calamus oil, chamomileoil, fir needle oil, garlic oil, coriander oil, carraway oil, lauriaetheroleum, lavender oil, lemon grass oil, lovage oil, bay oil, lupulistrobuli aetheroleum, mace oil, marjoram oil, mandarine oil, melissaoil, menthol, millefolii aetheroleum, mint oil, clary oil, nutmeg oil,spikenard oil, clove oil, neroli oil, niaouli, olibanum oil, ononidisaetheroleum, opopranax oil, orange oil, oregano oil, orthosiphon oil,patchouli oil, parsley oil, petit-grain oil, peppermint oil, tansy oil,rosewood oil, rose oil, rosemary oil, rue oil, sabinae aetheroleum,saffron oil, sage oil, sandalwood oil, sassafras oil, celery oil,mustard oil, serphylli aetheroleum, immortelle oil, fir oil, teatreeoil, terpentine oil, thyme oil, juniper oil, frankincense oil, hyssopoil, cedar wood oil, cinnamon oil, and cypress oil; and other oils suchas, for example, fish oil; and any analogs, derivatives, copolymers andcombinations thereof.

It should be appreciated that, in some embodiments, one of skill in theart may select one or more particular plasticizing agents in order toexclude any one or any combination of the above-described plasticizingagents.

In some embodiments, the plasticizing agent can include a component thatis water-soluble. In other embodiments, the plasticizing agent can bemodified to be water-soluble. In some embodiments, the plasticizingagent can include a component that is lipid-soluble. In otherembodiments, the plasticizing agent can be modified to be lipid-soluble.Any functional group can be added to modify the plasticizer's behaviorin a solvent such as, for example, body fluids that are present in vivo.

In a further aspect, the in vivo degradation or erosion rate of theplacental composition, as well as the density and cohesiveness of thecomponents, can be modified, for example, by cross-linking. Thecomponents can be homologously cross-linked and/or heterologouslycrosslinked. For example, the amnion can be cross-linked with itself,the intermediate tissue layer, chorion, a second amnion tissue and/or afiller. For example, a cross-linking agent can be added to thecomposition (e.g., amnion, chorion, intermediate tissue layer, filler,or any combination thereof as individual components and/or as tissuegrafts) prior to and/or after dehydration, micronization, and/or mixing.In general, the cross-linking agent is nontoxic and non-immunogenic.

As used herein, the term “homologously cross-linked” refers tocross-linking within one component of the amnion composition. Forexample, the amnion can be cross-linked with itself.

As used herein, the term “heterologously cross-linked” refers tocross-linking between different components of the placental composition.For example, the amnion can be cross-linked with the intermediate tissuelayer, chorion, a second amnion tissue and/or a filler.

When the amnion, intermediate tissue layer, chorion (or a tissue graftthereof), and/or filler are treated with the cross-linking agent, thecross-linking agent can be the same or different. In one aspect, theamnion, intermediate tissue layer, chorion, and/or filler can be treatedseparately with a cross-linking agent or, in the alternative, theamnion, intermediate tissue layer, chorion, and/or filler can be treatedtogether with the same cross-linking agent. In certain aspects, theamnion, intermediate tissue layer, chorion, and/or filler can be treatedwith two or more different cross-linking agents. The conditions fortreating the amnion, intermediate tissue layer, chorion, and filler canvary. In other aspects, the amnion, intermediate tissue layer, chorionand/or filler can be treated with a cross-linking agent before or aftermicronization. In one aspect, the concentration of the cross-linkingagent is from 0.1 M to 5 M, 0.1 M to 4 M, 0.1 M to 3 M, 0.1 M to 2 M, or0.1 M to 1 M. Preferably, the components are cross-linked prior todehydration such that the cross-linked components have a sufficientlylow water content to permit compression or molding in a non-porous mold.

In certain aspects, the placental composition can be treated with thecross-linking agent. Preferably, one or more components of the placentalcomposition are subjected to gas/fume cross-linking before or aftermicronization such that the water content of the component beingcross-linked is maintained at a low level, e.g., less than about 20%,less than about 15%, less than about 10%, or less than about 5%. Thecross-linking agent generally possesses two or more functional groupscapable of reacting with proteins to produce covalent bonds. In oneaspect, the cross-linking agent possesses groups that can react withamino groups present on the protein. Examples of such functional groupsinclude, but are not limited to, hydroxyl groups, substituted orunsubstituted amino groups, carboxyl groups, and aldehyde groups. In oneaspect, the cross-linker can be a dialdehyde such as, for example,glutaraldehyde. In another aspect, the cross-linker can be acarbodiimide such as, for example,(N-(3-dimethylaminopropyl)-N′-ethyl-carbodiimide (EDC). In otheraspects, the cross-linker can be an oxidized dextran, p-azidobenzoylhydrazide, N-[alpha-maleimidoacetoxy]succinimide ester, p-azidophenylglyoxal monohydrate, bis-[beta-(4-azidosalicylamido)ethyl]disulfide,bis-[sulfosuccinimidyl]suberate, dithiobis[succinimidyl]propionate,disuccinimidyl suberate, and1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride, abifunctional oxirane (OXR), ethylene glycol diglycidyl ether (EGDE),nordihydroguaiaretic acid (NDGA).

In one aspect, sugar is the cross-linking agent, where the sugar canreact with proteins present in the amnion, intermediate tissue layer,chorion, Wharton's jelly, and/or filler to form a covalent bond. Forexample, the sugar can react with proteins by the Maillard reaction,which is initiated by the nonenzymatic glycosylation of amino groups onproteins by reducing sugars and leads to the subsequent formation ofcovalent bonds. Examples of sugars useful as a cross-linking agentinclude, but are not limited to, D-ribose, glycerone, altrose, talose,ertheose, glucose, lyxose, mannose, xylose, gulose, arabinose, idose,allose, galactose, maltose, lactose, sucrose, cellibiose, gentibiose,melibiose, turanose, trehalose, isomaltose, or any combination thereof.

In one aspect, the chelating agent is a cross linking agent. In oneaspect, the chelating agent is substantially not involved incross-linking of the component(s).

In yet another embodiment of the invention, the metal ion incorporatedinto the composition is bound to a quinone group and/or a catechol grouppresent in the cross-linked placental composition. In other embodimentsof the invention, the metal ion incorporated into the composition may bebound to a basic nitrogen atom, non-limiting examples of which includeamino, or mono- or di-alkylated amino, and imidazole. In certainaspects, the metal ion is a platinum anticancer agent, such as platinum(+2) and/or platinum (+4). In certain preferred aspects, the platinumanticancer agent comprises a cis-diamino platinum moiety. As used hereinthe term “amino” refers to ammonia or a cycloalkyl amine, or aheteroaryl or a heterocyclyl amine, where the platinum coordinatingnitrogen atom can be a part of the ring system or derivatize the ringsystem. Examples of cis-diamino platinum moieties and other platinummoieties useful in this invention are described, e.g., in Kostova,Recent Patents on Anti-Cancer Drug Discovery, 2006, 1, 1-22, which isincorporated herein in its entirety by reference. In other aspects, theplatinum incorporated into the construct is present in an amount ofbetween about 0.1% to about 30%.

In certain aspects, the placental composition provides a sustainedrelease of metal ion, wherein the sustained release comprises aplurality of release rates including an immediate release, anintermediate release, an extended release or any combination of releaserates thereof. In other aspects, the plurality of release rates areadjusted to provide a suitable range of release rates, wherein the rangeof release rates comprises from about 1 minute to about 60 days, or anyrange therein.

The placental compositions can be formulated variously depending ontheir mode of delivery and their delivery site. In some embodiments, themicronized placental composition, such as amnion or chorion compositionsare compacted into a shape such as a pellet or an implant shaped as thegraphite tube in a pencil. Unit lengths or doses of such shaped soliddosage forms are also provided. Such solid forms of the compositions canbe administered topically to a site needing anticancer or othertreatment, or may be administered into a patient by using dry, solidinjection techniques well known and/or commercially available, or theirobvious modifications. Non-limiting examples of such solid injectionstechniques include, the Glide SDI, solid injection system.

In other embodiments, the placental compositions are formulated as aviscous fluid. Such viscous formulations may preferably includenon-aqueous organic liquids, which can include polymers such aspolyethylene glycols, Polaxamer® polymers, and the like, and/or smallmolecule organic solvents. Such viscous formulations may beadministered, preferably site specifically, using high pressure syringesthat are well known and/or commercially available, or obviousmodifications thereof. Non limiting examples of such high pressuresyringes include those described in U.S. Pat. No. 6,503,244(incorporated herein by reference) and the likes.

The present invention includes any combination of amnion, filler,plasticizer, and/or chelator, as would be understood by one of skill inthe art. The amnion and/or filler optionally is cross linked. In oneembodiment, the components are not cross linked. In an embodiment, theamnion and/or filler is homologously cross linked. In an embodiment, theamnion and/or filler is heterologously cross linked. The followingtables provide non-limiting examples of potential embodiments of theinvention. A person of skill in the art would understand suchembodiments may further comprise other components or aspects as providedherein.

Amnion Chelator Location Filler Plasticizer cross-linked amnion — − notcross-linked amnion — − cross-linked amnion cross-linked − notcross-linked amnion cross-linked − cross-linked amnion not cross-linked− not cross-linked amnion not cross-linked − cross-linked amnion — + notcross-linked amnion — + cross-linked amnion cross-linked + notcross-linked amnion cross-linked + cross-linked amnion notcross-linked + not cross-linked amnion not cross-linked + cross-linkedcollagen cross-linked − not cross-linked collagen cross-linked −cross-linked collagen not cross-linked − not cross-linked collagen notcross-linked − cross-linked collagen cross-linked + not cross-linkedcollagen cross-linked + cross-linked collagen not cross-linked + notcross-linked collagen not cross-linked + cross-linked plasticizer — +not cross-linked plasticizer — + cross-linked plasticizer cross-linked +not cross-linked plasticizer cross-linked + cross-linked plasticizer notcross-linked + not cross-linked plasticizer not cross-linked +cross-linked amnion + collagen cross-linked − not cross-linked amnion +collagen cross-linked − cross-linked amnion + collagen not cross-linked− not cross-linked amnion + collagen not cross-linked − cross-linkedamnion + collagen — + not cross-linked amnion + collagen — +cross-linked amnion + collagen cross-linked + not cross-linked amnion +collagen cross-linked + cross-linked amnion + collagen notcross-linked + not cross-linked amnion + collagen not cross-linked +cross-linked amnion + plasticizer — + not cross-linked amnion +plasticizer — + cross-linked amnion + plasticizer cross-linked + notcross-linked amnion + plasticizer cross-linked + cross-linked amnion +plasticizer not cross-linked + not cross-linked amnion + plasticizer notcross-linked + amnion + collagen + cross-linked plasticizercross-linked + amnion + collagen + not cross-linked plasticizercross-linked + amnion + collagen + cross-linked plasticizer notcross-linked + amnion + collagen + not cross-linked plasticizer notcross-linked + cross-linked chorion — − not cross-linked chorion — −cross-linked chorion cross-linked − not cross-linked chorioncross-linked − cross-linked chorion not cross-linked − not cross-linkedchorion not cross-linked − cross-linked chorion — + not cross-linkedchorion — + cross-linked chorion cross-linked + not cross-linked chorioncross-linked + cross-linked chorion not cross-linked + not cross-linkedchorion not cross-linked + cross-linked collagen cross-linked − notcross-linked collagen cross-linked − cross-linked collagen notcross-linked − not cross-linked collagen not cross-linked − cross-linkedcollagen cross-linked + not cross-linked collagen cross-linked +cross-linked collagen not cross-linked + not cross-linked collagen notcross-linked + cross-linked plasticizer — + not cross-linked plasticizer— + cross-linked plasticizer cross-linked + not cross-linked plasticizercross-linked + cross-linked plasticizer not cross-linked + notcross-linked plasticizer not cross-linked + cross-linked chorion +collagen cross-linked − not cross-linked chorion + collagen cross-linked− cross-linked chorion + collagen not cross-linked − not cross-linkedchorion + collagen not cross-linked − cross-linked chorion + collagen— + not cross-linked chorion + collagen — + cross-linked chorion +collagen cross-linked + not cross-linked chorion + collagencross-linked + cross-linked chorion + collagen not cross-linked + notcross-linked chorion + collagen not cross-linked + cross-linkedchorion + plasticizer — + not cross-linked chorion + plasticizer — +cross-linked chorion + plasticizer cross-linked + not cross-linkedchorion + plasticizer cross-linked + cross-linked chorion + plasticizernot cross-linked + not cross-linked chorion + plasticizer notcross-linked + chorion + collagen + cross-linked plasticizercross-linked + chorion + collagen + not cross-linked plasticizercross-linked + chorion + collagen + cross-linked plasticizer notcross-linked + chorion + collagen + not cross-linked plasticizer notcross-linked +

Another embodiment of the invention is directed to a method ofmanufacturing a placental composition, such as amnion or chorioncomposition comprising: providing an placental layer or placental tissuegraft; dehydrating the placental layer or graft; micronizing theplacental layer or graft; and binding one or more chelating agents tothe placental layer or graft, wherein the binding step may be before thedehydrating step or before or after the micronizing step.

Another embodiment of the invention is directed to a method ofmanufacturing a placental composition, such as amnion or chorioncomposition comprising: providing a placental layer or placental tissuegraft; dehydrating the placental layer or graft; micronizing theplacental layer or graft; binding one or more chelating agents to theplacental layer or graft, wherein the binding step may be before thedehydrating step or before or after the micronizing step; and chelatinga pharmacologically active metal ion to the composition, wherein thechelating step can be performed at any time after the binding step.

Another embodiment of the invention is directed to a method ofmanufacturing a placental composition, such as amnion or chorioncomposition comprising: providing a placental layer or placental tissuegraft and a filler; dehydrating the placental layer or graft andoptionally the filler; micronizing the placental layer or graft; andbinding one or more chelating agents to the placental layer or graftand/or to the filler, wherein the binding step may be before thedehydrating step or before or after the micronizing step; and combiningthe layer or graft and the filler, wherein the combination step may beperformed at any time during the process.

Another embodiment of the invention is directed to a method ofmanufacturing a placental composition, such as amnion or chorioncomposition comprising: providing a placental layer or placental tissuegraft and a filler; dehydrating the placental layer or graft andoptionally the filler; micronizing the placental layer or graft; bindingone or more chelating agents to the placental layer or graft and/or tothe filler, wherein the binding step may be before the dehydrating stepor before or after the micronizing step; combining the layer or graftand the filler, wherein the combination step may be performed at anytime during the process; and chelating a pharmacologically active metalion to the composition, wherein the chelating step can be performed atany time after the binding step.

The pharmaceutical compositions described herein can be administered ina number of ways depending on whether local or systemic treatment isdesired, and on the area to be treated. In one aspect, administrationcan be by injection. In other aspects, the composition can be formulatedto be applied internally to a subject. In other aspects, the compositioncan be applied topically, subdermally or subcutaneously.

III. Applications of Compositions Comprising Micronized Placental and/orFiller with One or More Chelating Agents Bound Thereto

Therapeutic Applications

The compositions comprising micronized placental compositions and/orfiller with one or more chelating agents bound thereto described hereinhave numerous therapeutic applications.

In one aspect, provided herein is a method of treating a subjectsuffering from cancer amenable to treatment with platinum alkylators,the method comprising administering placental composition in thesubject, wherein the composition comprises (a) micronized placental andone or more chelating moieties and (b) platinum chelated to thechelating moieties, to administer an effective amount of platinum intothe subject. The placental composition optionally comprises a filler.The chelating moieties may be bound to the amnion, filler, or both.

In yet another embodiment, the invention is directed to a method oftreating a subject in need of treatment for a cancer treatable with ametal-containing anticancer agent, comprising: a) implanting a medicalconstruct in a subject, wherein the medical construct comprises amicronized placental composition and an anticancer amount of ananticancer agent incorporated therein to provide a therapeuticallyeffective amount of anticancer platinum in the construct, and b)releasing the anticancer agent from the composition, thereby inhibitingcancer. In one embodiment, the effective amount of anticancer agent,e.g. platinum, is released from the composition at a plurality of invivo release rates. Methods of determining the therapeutically effectiveamount and/or appropriate mode of administration of the compounds andcompositions provided herein will be apparent to the skilled artisanupon reading this disclosure and based on other methods known to them.In one embodiment, the placental composition optionally comprises afiller.

The present invention finds use in medical applications and animalstudies. The term “medical” includes both human and veterinary uses.Suitable subjects of the present invention include, but are not limitedto avians and mammals.

In particular embodiments, the subject is “in need of” the methods ofthe present invention, e.g., the subject may benefit from a surgicalprocedure implanting a composition of the present invention, such as aprosthesis or other device. In certain embodiments, after implantation,the compositions of the present invention can confer a therapeuticand/or prophylactic effect to the subject, such as prevent a diseaseand/or clinical symptom, reduce the severity of a disease and/orclinical symptom relative to what would occur in the absence of themethods of the prevent invention, and/or delay the onset and/orprogression of a disease and/or clinical symptom. The methods of thepresent invention can provide complete and/or partial treatment and/orprotection. In particular embodiments, after implantation in oradministration to a subject, the compositions of the present inventiontreat and/or inhibit and/or protect against cancer, preferably thosecancers that are treatable with platinum anticancer agents, in thesubject.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how thecompounds, compositions, and methods described and claimed herein aremade and evaluated, and are intended to be purely exemplary and are notintended to limit the scope of what the inventors regard as theirinvention. Efforts have been made to ensure accuracy with respect tonumbers (e.g., amounts, temperature, etc.) but some errors anddeviations should be accounted for. Unless indicated otherwise, partsare parts by weight, temperature is in ° C. or is at ambienttemperature, and pressure is at or near atmospheric. There are numerousvariations and combinations of reaction conditions, e.g., componentconcentrations, desired solvents, solvent mixtures, temperatures,pressures and other reaction ranges and conditions that can be used tooptimize the product purity and yield obtained from the describedprocess. Only reasonable and routine experimentation will be required tooptimize such process conditions.

Example 1 Preparation of Micronized Composition

Amnion/chorion tissue grafts used here to produce the micronizedparticles were produced by the process described in US 2008/0046095,which is incorporated by reference in its entirety. Tissue grafts (4cm×3 cm) and two 9.5 mm steel grinding balls were placed in 50 mL vialsand the vials subsequently sealed. The vials were placed in theCryo-block, and the Cryo-block was placed in a Cryo-rack. The Cryo-rackwas placed into a liquid nitrogen holding Dewar. Tissue samples weresubjected to vapor phase cooling for no more than 30-60 minutes. TheCryo-rack was removed from the Dewar, and the Cryo-block was removedfrom the Cryo-rack. The Cryo-block was placed into the Grinder (SPEXSample Prep GenoGrinder 2010) and set at 1,500 rpm for 20 minutes. After20 minutes has elapsed, the tissue is inspected to ensure micronization.If necessary, the tissue can be placed back into the Dewar for anadditional 30-60 minutes, and moved to the grinder for an additional 20minutes to ensure sufficient micronization. Once the tissue issufficiently micronized it is sorted using a series of American StandardASTM sieves. The sieves were placed in the following order: 355 μm, 300μm, 250 μm, 150 μm, and 125 nm. The micronized material was transferredfrom the 50 mL vials to the 355 μm sieve. Each sieve was agitatedindividually in order to thoroughly separate the micronized particles.Once the micronized particles have been effectively separated using thesieves, the micronized particles having particle sizes of 355 μm, 300μm, 250 μm, 150 μm, and 125 μm were collected in separate vials.

Example 2 Preparation of Tissue Grafts with Micronized Placental Tissue

Various modifications and variations can be made to the compounds,compositions and methods described herein. Other aspects of thecompounds, compositions and methods described herein will be apparentfrom consideration of the specification and practice of the compounds,compositions and methods disclosed herein. It is intended that thespecification and examples be considered as exemplary.

A detailed description of reinforced placental tissue grafts is providedin U.S. Patent Application Publication No. 2014/0067058 whichapplication is incorporated herein by reference in its entirety.

A detailed description of making and using micronized placental tissueand extracts thereof is provided in U.S. Patent Application PublicationNo. 2014/0050788 which application is incorporated herein by referencein its entirety.

Example 3 Cell Migration in the Presence of EpiFix®

Human mesenchymal stem cells (human MSC) were evaluated in cell culturein the presence of samples of EpiFix® to determine whether the EpiFix®would induce migration of the human MSC. EpiFix® is a layer of amnionand chorion with the epithelial layer intact.

Materials and Methods

Standard migration assays were performed in 24-well cell culture insertswith 8-μm pore membrane filters at the bottom of the insert (see FIG. 4;BD Biosciences). 24 hours prior to the start of the experiment, humanMSCs (one donor, passage 3) were cultured in serum free media, and 300μL of 5 μg/mL fibronectin in PBS was placed into each cell cultureinsert to enable adsorption of fibronectin to the cell culture insertsurface overnight.

On the day of the experiment, 700 μL of serum-free culture medium wasloaded into the bottom wells of the plate, followed by the addition ofdifferently sized portions of sterilized EpiFix® (Low: 1.5-mm diameterdisk; Medium: 4-mm diameter disk; High: 12×13 mm square, trimmed into3-4 mm square pieces; n=6 EpiFix® tissue donors tested). One squarecentimeter of EpiFix® weighs 4 mg. Serum-free medium and medium with 10%fetal bovine serum (n=6) acted as negative and positive controls,respectively. Human MSCs (40,000 cells in 300 μL) were then loaded intothe cell culture inserts and cultured for 24 hours. Then, both sides ofthe cell culture inserts were rinsed with PBS, and non-migrating cellsin the upper portion insert were removed with a cotton-tippedapplicator. Cells on the lower side of the insert plus the membranefilter were fixed in 10% formalin for 20 minutes, then rinsed andstained with hematoxylin for 5 min. The number of cells migratingthrough the membrane were counted on the lower surface of the membranewith an inverted microscope (Nikon TE2000; SPOT Software 4.6).

Data were normalized to the 10% FBS positive control and are expressedas mean±standard deviation of counted, migrated cells per 100× fieldmicrograph for each sample well. Statistical comparisons were performedusing a Box-Cox transformation to normalize data variance, followed byone-factor analysis of variance (ANOVA) with Tukey's honestlysignificant difference post-hoc test.

Results

The Low group (1.5 mm diameter disk) containing the smallest EpiFix®sample was not significantly different from the no serum negativecontrol (see bar graph in FIG. 5). Both the Medium group (4 mm diameterdisk) and the High group (12×13 mm square, trimmed into 3-4 mm squarepieces) were statistically higher than the no serum control (about 60%and 75% migration relative to control; see FIG. 5), indicating thatEpiFix® stimulated cell migration. The High group was not significantlydifferent from the Medium group. The results indicate that the EpiFix®product contains one or more factors that attract human mesenchymal stemcells.

Example 4 Stem Cell Recruitment in Mice Receiving EpiFix® Implants

A study was undertaken to determine whether EpiFix® implanted in normalmice causes recruitment of stem/progenitor cells, focusing on mousehematopoietic stem cells (HSCs) and mouse mesenchymal stem cells (mouseMSCs).

Materials and Methods

EpiFix® products from six donors were used for implantation in normalmice. A 5×5 mm square of EpiFix® was surgically placed subcutaneously in4 month old FVB/NJ mice (weighing between about 23.50 g and about 30 g).Four mice were implanted per sample per time point. The time points were3, 7, 14 and 28 days. The negative controls were normal skin and shamoperated mice (surgical incision but no implant). Decellularized dermalmatrix (acellular dermal matrix; ADM) was used as the comparativeimplant (Type I collagen, no cytokines). The implant and overlying skinwas harvested for fluorescence-activated cell sorting (FACS).

Implants and overlying skin were harvested, cut into 1 mm² sections, andincubated in a 0.15% dispase/0.075% collagenase solution at 37° C. for 1hour. After centrifugation, samples were stained with a lineage antibodycocktail as described below. CD31 antibody was added followed by AlexaFluor 647 anti-rat secondary antibody. Phycoerythrin-Cy7-conjugatedanti-CD45 antibody was incubated last. Samples were prepared andanalyzed as described below.

Samples were incubated with a lineage negative (lin⁻) antibody cocktail(Ter119/CD4/CD8a/Gr-1/CD45R/CD11b) followed by phycoerythrin-Cy5anti-rat secondary antibody. For mesenchymal stem cell analysis,conjugated antibodies were added against CD45 (phycoerythrin-Cy7) andSca-1 (fluorescein isothiocyanate). For hematopoietic stem cellanalysis, conjugated antibodies were added against CD45(phycoerythrin-Cy7), c-Kit (phycoerythrin), and Sca-1 (fluoresceinisothiocyanate). Samples were incubated with antibodies for 30 minutesand then washed by adding 5 volumes of 2% fetal bovine serum inphosphate-buffered saline with 2 mM ethylenediaminetetraacetic acid.Cells were centrifuged and then re-suspended in propidium iodide for 1minute at 4° C. Samples were analyzed using an LSR Flow Cytometer. UsingCellQuest software), samples were gated for lin⁻/Sca-1⁺/CD45⁻ to definemesenchymal stem cells and for lin⁻/Sca-1⁺/c-Kit⁺/CD45⁺ to definehematopoietic stem cells.

Results

Mouse HSCs were significantly increased following EpiFix® implantationcompared to negative controls at days 7, 14 and 28 (see FIG. 6A). MouseHSCs remained significantly increased in the EpiFix® samples at day 28compared to ADM.

Mouse MSCs were significantly increased following EpiFix® implantationcompared to negative controls at day 7 (see FIG. 6B). The averagepercentages of mouse MSCs were increased at all time points compared tonegative controls.

Thus the data described above show that EpiFix® implants effectivelyrecruit both HSCs and MSCs in vivo in normal mice. The data also showthat EpiFix® leads to longer term HSC recruitment than acellular dermalmatrix (ADM), supporting the hypothesis of a cytokine mediated effect ofEpiFix®.

Example 5 Stem Cell Characterization in Mice Receiving EpiFix® Implants

A study was undertaken to characterize stem cells recruited to EpiFix®implantation sites in mice, using flow cytometry andimmunohistochemistry.

Materials and Methods

Sterile, Purion® processed EpiFix® in a 5×5 mm square patch wasimplanted subcutaneously through a skin incision on the backs of sixteen4 month old FVB/NJ mice. Identical skin incisions were made in anothersixteen mice to function as a control treatment (sham). For comparisonwith a collagen scaffold, a 5×5 mm square patch of decellularized humandermis (acellular dermal matrix; ADM) was implanted subcutaneously onthe backs of sixteen mice. Un-operated mice were used as a source of“normal” back skin for the analyses.

The surgical site was removed at 3, 7, 14 and 28 days followingimplantation for analyses of stem cells. Four animals/group were used ateach time point. Stem cells were identified with two distinct methods:Fluorescence-activated cell sorting (FACS) and immunohistochemistry(IHC). For the FACS analysis, all cells were isolated from the amnionand associated regenerated tissue. The cells were fluorescently labeledwith antibodies to specific stem cell markers. The identity and numberof each cell type were determined with a flow cytometer.

For the immunohistochemical analyses, the membrane and associatedregenerated tissue was fixed, sectioned for slides, and stained withspecific antibodies to stem cells. Two antibodies were used for theimmunohistochemistry: anti-CD34, which specifically detectshematopoietic progenitor cells (HPC), and reacts with dermal progenitorcells, endothelial cells, dendritic cells; and anti-CD31, which detectsendothelial cells. The stained tissue sections were examinedmicroscopically and the presence and number of specific stem cell typeswere measured. For the experimental analysis, the relative number ofeach cell type was counted. The results were calculated as thepercentage of each cell type (no. of immunostained cells/total number ofcells). Two areas were analyzed immunohistochemically for cellrecruitment: the tissue surrounding the implant and the implant itself

Results

Representative data from the FACS analyses are shown in FIG. 7A. Theleft panel shows the total number of cells in the sample. The middlepanel shows the number of CD45 positive cells (in inset box). The rightpanel shows the number of Sca-1 positive cells (in inset box). CD45 andSca-1 are specific markers for hematopoietic stem cells.

FIG. 7B shows an exemplary immunohistochemistry image. The gray bar inthe lower left corner represents 50 μm. The section was stained withDAPI (blue—stains all cells) and anti-CD34 (red). The place where thetissue is implanted in the experimental mice is shown for reference.

Hematopoietic progenitor cell (HPC) levels were significantly elevatedin tissue surrounding EpiFix® implants at days 14 and 28 compared tonegative controls. Hematopoietic progenitor cells were significantlyincreased in the tissue surrounding the EpiFix® implant at days 14 and28 compared to collagen scaffold ADM control.

Progenitor cells were recruited into the EpiFix® implant. Intra-implanthematopoietic progenitor cells peaked at day 14 in the EpiFix® implant,and remained elevated at day 28. Average intra-implant hematopoieticprogenitor cells were increased in the EpiFix® implant at days 14 and 28compared to control ADM. Progenitor cells were not recruited into theADM control implant.

Vascularization of the EpiFix® implant steadily increased from day 14 today 28. The amount of new vessel formation in the EpiFix® implant wassignificantly greater than that in the ADM control on day 28.

These data establish that EpiFix® contains one or more factors thatrecruit both hematopoietic stem cells and mesenchymal stem cells to thesite of injury. More of these stem cells were found in the EpiFix®membrane and associated regenerated tissue than in the sham or, moreimportantly, the control collagen scaffold. EpiFix® was significantlymore effective than the control decellularized collagen scaffold inrecruiting progenitor cells to colonize the implant site. There weremore progenitor cells in the EpiFix® membrane than in the controlcollagen scaffold.

EpiFix® also induced new blood vessel formation in the associatedregenerated tissue and the EpiFix® membrane itself. Vascularization inthe EpiFix® membrane was significantly higher than in the collagenscaffold control.

Example 6 Preparation of Non-Cross-Linked Tissue-Chelator Conjugates

Various placental tissue grafts described above can be combined with atleast 30 equivalents of dopamine under conventional amide formingconditions to provide for a plurality of chelating moieties bound to theplacental tissue. The nitrogen of the dopamine reacts with a number ofcarboxylic acid groups of the amniotic collagen to form a carbodiamidelinkage.

Example 7 Chelation of the Biologically Active Metal Ion with thePolymer Chelator Conjugate

Dry non-cross-linked tissue-chelator conjugates described above areincubated in 1% (w/v) cis-diamino Pt(II) diaqua salt in water at roomtemperature for 16 hours. The grafts are then washed with deionizedwater and dried. Platinum content in the conjugates is measured e.g., byInductively Coupled Plasma-Mass Spectrometry (ICP-MS). Results areexpressed as μg/g, which is equivalent to parts per million (ppm).

The incorporation of platinum in the conjugates can produce abiomaterial imbibed with anticancer capabilities. As such, the method ofincorporating platinum into tissue materials can provide a drug deliverydevice, preferably for anticancer drug delivery.

In some embodiments, the tissues utilized in the conjugates of thepresent invention also incorporate one or more stem cell recruitingfactors that enhance stem cell chemotaxis and/or recruitment. Suchcompositions for recruiting stem cells are described in U.S. PatentApplication Publication No. 2014/0106447, which application isincorporated herein by reference in its entirety. These stem cellrecruiting factors in combination with the biologically active metal areabhorrent to aberrant stem cells, such as cancerous cells, and workagainst tumor-related recurrence of cancer.

It is evident from the above examples that the EpiFix® amniotic membraneallograft has the capability to attract or increase the flux of stemcells to the amnion. Thus, the amniotic membrane is a biologicallyderived polymer which attracts stem cells. Further, if the amnioticmembrane is conjugated to the chelator moiety and a metal such as e.g.,cisplatin, then the polymer conjugate can be used to kill stem cells,particularly aberrant stem cells such as tumor cells.

Various modifications and variations can be made to the compounds,compositions and methods described herein. Other aspects of thecompounds, compositions and methods described herein will be apparentfrom consideration of the specification and practice of the compounds,compositions and methods disclosed herein. It is intended that thespecification and examples be considered as exemplary.

A detailed description of suitable cross-linking agents and proceduresis provided in U.S. Patent Application Publication No. 2014/0052247which application is incorporated herein by reference in its entirety.

A detailed description of micronized placental tissue is provided inU.S. Patent Application Publication Nos. 2014/0052274 and 2014/0050788,which applications are incorporated herein by reference in theirentireties.

A detailed description of cross-linked polymers comprising metal ions isprovided in U.S. Patent Application Publication Nos. 2014/0141096,2014/0142041, and U.S. patent application Ser. No. 13/860,473, all ofwhich applications are incorporated herein by reference in theirentireties.

What is claimed:
 1. A composition comprising i) a micronized placentaltissue component; ii) one or more chelating moieties; and iii)optionally, a biologically compatible filler; wherein the one or morechelating moieties are covalently bound to the placental tissuecomponent and/or the filler, when the filler is present; and wherein atherapeutically effective amount of a pharmacologically active metal ionis chelated to the one or more chelating moieties.
 2. The composition ofclaim 1, wherein the placental tissue component and/or filler ishomologously cross-linked.
 3. The composition of claim 1, wherein theplacental tissue component and/or filler is heterologously cross-linked.4. The composition of claim 1, wherein the filler is present in saidcomposition and is a biologically compatible polymer.
 5. The compositionof claim 4, wherein the biologically compatible polymer is selected fromthe group consisting of collagen, hyaluronic acid, and a plasticizer. 6.The composition of claim 1, wherein the ratio of micronized placentaltissue component to filler is between 1:99 and 99:1.
 7. The compositionof claim 1, wherein one or more of the chelating moieties cross-link theplacental tissue component and/or the filler.
 8. The composition ofclaim 1, wherein the one or more chelating moieties do not cross-linkthe placental tissue component.
 9. The composition of claim 1, furthercomprising a pharmaceutically acceptable excipient.
 10. The compositionof claim 1, wherein the pharmacologically active metal ion is in ananticancer agent.
 11. The composition of claim 10, wherein theanticancer agent comprises a platinum ion.
 12. The composition of claim11, wherein the anticancer agent is cisplatin.
 13. The composition ofclaim 1, wherein the composition provides a sustained release of themetal ion.
 14. The composition of claim 1, wherein the composition ismolded.
 15. The composition of claim 1, wherein the micronized placentaltissue component is micronized amnion.
 16. The composition of claim 15,further comprising micronized chorion, Wharton's jelly, and/ormicronized intermediate tissue layer.
 17. The composition of claim 1,wherein the micronized placental tissue component is micronized chorion.18. A composition comprising i) micronized amnion; ii) a biologicallycompatible filler; iii) a biologically compatible plasticizer; and iv)one or more chelating moieties; wherein the one or more chelatingmoieties are covalently bound to the micronized amnion and/or thefiller; and wherein a therapeutically effective amount of apharmacologically active metal ion is chelated to the one or morechelating moieties.
 19. The composition of claim 1, wherein the fillercomprises bone particles.